# Afferent-driven modulation of spinal interneuron circuits across disease stages in amyotrophic lateral sclerosis

Sina Sangari, Alexandra Lackmy-Vallée, Iseline Peyre, Pierre-François Pradat, Véronique Marchand-Pauvert

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Sina Sangari, Alexandra Lackmy-Vallée, Iseline Peyre, Pierre-François Pradat, Véronique Marchand-Pauvert. Afferent-driven modulation of spinal interneuron circuits across disease stages in amyotrophic lateral sclerosis. Neurobiology of Disease, 2026, 219, pp.107270. 10.1016/j.nbd.2026.107270. hal-05460777

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# Afferent-driven modulation of spinal interneuron circuits across disease stages in amyotrophic lateral sclerosis

Sina Sangari<sup>a,b,c,1</sup>, Alexandra Lackmy-Vallée<sup>a</sup>, Iseline Peyre<sup>a</sup>, Pierre-François Pradat<sup>a,d</sup>, Véronique Marchand-Pauvert<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

- Amyotrophic lateral sclerosis
- Spinal cord
- Inhibitory drive
- Excitability
- Sensorimotor integration
- Neural disinhibition
- Disease progression

## ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that progressively disrupts voluntary motor command through combined cortical and spinal motor neuron degeneration. However, how spinal circuits reorganize during the disease remains poorly understood, particularly in humans. This study examined the function of excitatory and inhibitory spinal interneuron circuits that control upper and lower limb movements, using non-invasive electrophysiological techniques targeting specific afferent-motoneuron pathways at cervical and lumbar levels. These segments are clinically relevant, as spinal-onset forms constitute the predominant clinical presentation of ALS. We compared patients with ALS at different stages of functional impairment to healthy individuals. Spinal circuits predominantly driven by muscle spindle afferents (group Ia and II) showed, at the group level, a marked reduction in inhibition together with enhanced propriospinal excitation. In contrast, pathways mediated by tendon afferents (group Ib) and cutaneous inputs appeared preserved in unstratified analyses. However, when accounting for disease stage, inhibitory dysfunction emerged as an early feature, whereas excitation increased progressively with functional impairment, and modulations also became detectable in Ib- and cutaneous-driven responses. These findings reveal an afferent- and stage-dependent hierarchy of spinal dysfunction, following a reproducible sequence from early disinhibition to maladaptive excitation. This dynamic pattern mirrors the organization observed in preclinical spinal models and aligns with cortical pathophysiology, where widespread loss of inhibition precedes selective increases in excitation. Together, these results refine the mechanistic understanding of motor network disorganization in ALS and identify inhibitory interneurons as potential therapeutic targets to stabilize spinal network function.

## 1. Introduction

Amyotrophic lateral sclerosis (ALS) is a multisystem neurodegenerative disorder characterized by the progressive loss of layer V pyramidal cells in the brain motor cortex (upper motor neurons, UMNs) and of bulbo-spinal motoneurons that connect to muscle fibers (lower motor neurons, LMNs). This combined degeneration leads to progressive paralysis and death within a few years of symptom onset (González-Sánchez et al., 2025). Beyond the selective vulnerability of motor neurons, growing evidence supports a network-driven view of ALS pathogenesis, in which circuit-level dysfunction, rather than isolated neuronal

loss, plays a central role (Salzinger et al., 2024). Neural hyperexcitability has emerged as a hallmark feature of the disease, consistently reported across both animal models and human studies, even at pre-symptomatic and early clinical stages (Eisen et al., 2023). At the spinal level, accumulating evidence indicates that motoneurons (LMNs) themselves are not intrinsically hyperexcitable (Delestrée et al., 2014; Devlin et al., 2015; Marchand-Pauvert et al., 2019). Instead, abnormal spinal excitability likely results from a disruption in the balance of inhibitory and excitatory inputs they receive, mirroring changes at the cortical level, where early interneuron dysfunction leads to disinhibition (Goffin et al., 2024; Higashihara et al., 2024; Montañana-Rosell et al.,

<footnote><sup>a</sup> Sorbonne Université, INSERM, CNRS, Laboratoire d'Imagerie Biomédicale, LIB, F-75006 Paris, France</footnote>

<footnote><sup>b</sup> Shirley Ryan AbilityLab, Chicago, IL 60611, United States of America</footnote>

<footnote><sup>c</sup> Department of Physical Medicine and Rehabilitation, Northwestern University, Chicago, IL 60611, United States of America</footnote>

<footnote><sup>d</sup> AP-HP, Hôpital Pitié-Salpetrière, Maladies du Système Nerveux, F-75013 Paris, France</footnote>

<footnote>* Corresponding author at: Laboratoire d'Imagerie Biomédicale, Sorbonne Université Campus des Cordeliers, 15 rue de l'École de Médecine, 75006 Paris, France. E-mail address: veronique.marchand-pauvert@inserm.fr (V. Marchand-Pauvert).</footnote>

<footnote n="1">¹ Equally involved</footnote>
2024; Nascimento et al., 2024). This paradigm shift has brought spinal interneurons into focus as key players in ALS pathophysiology.

Spinal interneurons shape motor output by integrating sensory and descending inputs within highly organized sensorimotor networks (Pierrot-Deseilligny and Burke, 2005). In ALS mouse models, inhibitory interneurons, such as group Ia inhibitory interneurons mediating reciprocal inhibition between antagonistic muscle pairs and Renshaw cells mediating recurrent inhibition (genetically identified as V1 interneurons), show early degeneration that precedes or parallels motoneuron loss. Excitatory interneurons, including V2a glutamatergic subtypes, appear more resistant initially but also undergo functional remodeling and degeneration (Goffin et al., 2024; Montañana-Rosell et al., 2024; Nascimento et al., 2024). Collectively, these findings suggest that ALS involves a dynamic disruption of spinal microcircuits, following a characteristic temporal sequence: early disinhibition followed by maladaptive excitation. Similar temporal patterns have been reported at the cortical level in humans using transcranial magnetic stimulation (TMS) in humans (Pavey et al., 2024), yet spinal circuitry in ALS patients remains far less explored.

Several electrophysiological paradigms combining electromyography (EMG) with peripheral nerve stimulation and TMS allow indirect probing of spinal microcircuits in humans, targeting both inhibitory and excitatory interneurons (Pierrot-Deseilligny and Burke, 2012; Pierrot-Deseilligny and Burke, 2005). Although these techniques provide only indirect evidence, assessing motoneuronal output through EMG modulations, they have been extensively validated as robust proxies for specific spinal pathways. Despite their potential, only a few studies have applied these approaches systematically in ALS. Most have focused on the soleus Hoffmann (H)-reflex, thereby restricting current knowledge of spinal circuit dysfunction to the lumbar cord and to a motoneuron pool that is not among the earliest to degenerate (Castro et al., 2024; Castro et al., 2021; Howells et al., 2020; Mazzocchio and Rossi, 2010; Özyurt et al., 2020; Sangari et al., 2022; Schieppati et al., 1985). This narrow focus limits our understanding of how spinal dysfunction evolves across different levels (cervical vs. lumbar) and interneuron subtypes (inhibitory vs. excitatory). There is therefore a critical need to comprehensively characterize spinal circuit dysfunction in ALS, not only to refine our understanding of human disease mechanisms but also to assess the translational relevance of preclinical findings (Montañana-Rosell et al., 2024). Demonstrating convergence between human and animal data provides strong validation for murine models and supports their use to explore mechanisms inaccessible in patients.

In this study, we investigated the integrity of spinal interneuronal circuits in a large cohort of ALS patients compared with age- and sex-matched healthy controls, using a comprehensive electrophysiological approach based on EMG recordings. We applied a set of paradigms specifically designed to assess inhibitory and excitatory spinal pathways at both cervical and lumbar levels. Our goal was to determine whether ALS is associated with reproducible patterns of dysfunction across microcircuits, whether these alterations differ between spinal segments, and whether they follow a structured temporal progression related to disease severity or phenotype. Clarifying these points may provide new insights into the role of spinal interneuronal networks in ALS pathophysiology and guide the development of targeted therapeutic strategies.

# 2. Materials and methods

## 2.1. Experimental design

This study aimed to characterize the spinal excitatory and inhibitory circuit alterations in patients with ALS and to examine their relationships with clinical status. Neurophysiological recordings were obtained from a cohort of ALS patients, and age- and sex-matched healthy controls using standardized stimulation protocols. The data were analyzed to compare group-level effects and to classify individual responses based

on deviations from control ranges. Clinical variables, including functional impairment and disease stage, were integrated post hoc to assess their associations with circuit-level changes using both univariate and multivariate approaches. Analyses followed a predefined structure, beginning with group comparisons, followed by response classification and association with clinical features.

## 2.2. Ethics

This study is part of a broader investigation into spinal excitability in ALS patients (SpinalBioMark-SLA), a research program in which multiple spinal circuitries have been assessed using indirect electrophysiological techniques. Earlier analyses from this cohort reported that LMNs are not intrinsically hyperexcitable, that recurrent inhibition declines with disease progression, and that Hoffman (H)-reflex transmission is altered across different muscle groups (Marchand-Pauvert et al., 2019; Sangari et al., 2024; Sangari et al., 2022). The present study builds on this framework by examining additional spinal interneuron circuits and reports entirely original, previously unpublished findings regarding afferent-driven excitatory and inhibitory pathways at both cervical and lumbar levels.

The experimental procedures adhere to the latest revision of the World Medical Association's Code of Ethics (Declaration of Helsinki) and were approved by the Inserm Ethics Committee (protocol no. C14-21) as well as the national ethical authorities (CPP Ile-de-France, Paris 6 - Pitié-Salpêtrière, CPP/16-15; RCB 2014-201-A01240-47). The study is registered in a public database (https://clinicaltrials.gov, NCT02429492) (Pradat and Marchand-Pauvert, 2015). Written informed consent was obtained from all participants prior to the experiments.

## 2.3. Participants

The protocol aimed to test cervical and lumbar interneurons. For each spinal circuitry, sample sizes were predetermined using data from previous studies on healthy controls conducted in the laboratory. These calculations, performed with StatEL (ad Science, Paris, France), were based on a first-type error risk of 5%, a second-type error risk of 10%, and assumed a minimum difference of 10% between control and ALS groups. A total of 58 patients (13 women; mean age ± 1 standard deviation [SD]: 62.8 ± 9.7 years) and 59 controls (13 women; 62.4 ± 9.2 years) were included. According to the sample size estimates, not all participants completed every experiment designed to assess the different spinal circuitries. Moreover, two patients and one control did not exhibit the investigated responses in the upper and/or lower limbs. The number of participants included in each experiment, along with the reasons for exclusions, are provided in Table S1 in Supplementary materials.

The main inclusion criteria (Pradat and Marchand-Pauvert, 2015) included i) for controls, the absence of prior or current neurological illness, and ii) for patients, probable or definite ALS according to the El Escorial criteria (Brooks et al., 2000), no or weak motor deficit in the proximal muscles (arm and thigh muscles) as determined by manual muscle testing and scored using cumulative Medical Research Council (MRC) ratings (Table S2, Supplementary materials), together with the presence of distal upper and/or lower limb involvement, and no peripheral neuropathy and no comorbid neurological conditions. These criteria were required to investigate heteronymous spinal projections, which in humans follow a distal-to-proximal organization in both upper and lower limbs (Pierrot-Deseilligny and Burke, 2012; Pierrot-Deseilligny and Burke, 2005), by probing afferent inputs from distal muscles onto relatively preserved proximal motor pools, and inherently limited the inclusion of patients with purely bulbar presentations as well as flail arm phenotypes. Patients were screened and tested for the four most common ALS-causing mutations (SOD1, FUS, C9orf72 and TDP43). All were negative except one (C9orf72, patient #11; Supplementary Table S2). All participants were preferably tested on their dominant side
(Oldfield, 1971). However, to ensure accurate interpretation of EMG results, which can be significantly influenced by ALS-related peripheral denervation (Pierrot-Deseilligny and Burke, 2012; Vucic et al., 2024), patients were assessed on their less affected side for the target motor nuclei examined in each experiment. When motor deficits were observed on the dominant side, the non-dominant side was tested instead. Additionally, in both groups, the non-dominant side was investigated in cases of orthopedic trauma affecting the dominant side. A total of 45/56 patients and 56/58 controls were tested on the dominant side.

The main demographic and clinical characteristics of early- and late stage are summarized in Table 1, with detailed individual clinical data provided in Supplementary Table S2. Disease stage was defined based on the typical diagnostic delay in ALS, which generally ranges from 12 to 18 months (Mitchell et al., 2010; Richards et al., 2020). Patients assessed within 18 months of symptom onset were classified as early stage, whereas those evaluated after 18 months were classified as late stage. The mean disease progression rate was calculated as the mean number of ALSFRS-r points lost per month (48 – ALSFRS-r score / disease duration). Functional impairment was defined according to ALSFRS-r scores (Supplementary Table S2): patients scoring ≥41 were classified as weakly impaired, those scoring 34–40 as moderately impaired, and those scoring ≤33 as severely impaired (Genge et al., 2025; Miller et al., 2022). The predominant form was determined based on neurological examination, taking into account signs of central involvement (Babinski and Hoffman signs, spasticity and hyperreflexia) and peripheral involvement (muscle strength, atrophy and fasciculation).

<caption>**Table 1**</caption>

Demographic and main clinical features of patients stratified by disease stage. Key demographic and clinical variables are reported for patients classified as early- or late-stage ALS (cut-off = 18 months since first symptoms). Age and disease duration are presented as mean ± SD, ALSFRS-r, as median [range], and categorical features as number of patients in each subgroup (percentage). The distribution column reports the statistical comparisons between groups (Early vs. Late stage), with Student's t-tests used for continuous variables and $Chi^2$ tests for categorical variables. Abbreviations: B, bulbar; UL, upper limb; LL, lower limb; Dom, handedness-related dominant side; N-Dom, handedness-related non-dominant side; UMN, upper motor neuron; LMN, lower motor neuron; ALSFRS-r, revised ALS Functional Rating Scale.

<table><thead><tr><th></th><th>Early stage (32/56 patients)</th><th>Late stage (24/56 patients)</th><th>Distribution</th></tr></thead><tbody><tr><td>Gender (F/M)</td><td>9/23 (28.1 / 71.2%)</td><td>4/20 (16.7 / 83.3%)</td><td>Chi<sup>2</sup> = 1.01<br>P = 0.31</td></tr><tr><td>Age (years)</td><td>63.0 ± 9.6</td><td>61.7 ± 9.8</td><td>t = -0.52<br>P = 0.61</td></tr><tr><td>Onset site (B/UL/LL)</td><td>6/13/13 (18.8 / 40.6 / 40.6%)</td><td>4/8/12 (16.7 / 33.3 / 50.0%)</td><td>Chi<sup>2</sup> = 0.50<br>P = 0.78</td></tr><tr><td>Side of onset (Dom/N-Dom)</td><td>13/13 (50 / 50%)</td><td>9/11 (45 / 55%)</td><td>Chi<sup>2</sup> = 0.15<br>P = 0.93</td></tr><tr><td>Disease duration (months)</td><td>10.5 ± 3.8</td><td>34.5 ± 17.0</td><td>t = 6.77<br>P < 0.001</td></tr><tr><td>ALSFRS-r (median [range])</td><td>42 [24–47]</td><td>40 [34–45]</td><td>t = -0.44<br>P = 0.66</td></tr><tr><td>Mean progression rate (slow/fast)</td><td>10/22 (31.2 / 68.8%)</td><td>20/4 (83.3 / 16.7%)</td><td>Chi<sup>2</sup> = 15.0</td></tr><tr><td>Impairment (weak/moderate/severe)</td><td>18/9/8 (56.2 / 28.1 / 15.7%)</td><td>8/16/0 (33.3 / 66.7 / 0%)</td><td>Chi<sup>2</sup> = 9.86<br>P < 0.01</td></tr><tr><td>Predominant form (UMN/LMN/Mixed)</td><td>19/7/6 (59.4 / 21.9 / 18.7%)</td><td>11/6/7 (45.8 / 25.0 / 29.2%)</td><td>Chi<sup>2</sup> = 1.17<br>P = 0.56</td></tr><tr><td>Spasticity (yes/no)</td><td>8/24 (25 / 75%)</td><td>5/19 (20.8 / 79.2%)</td><td>Chi<sup>2</sup> = 0.13<br>P = 0.71</td></tr><tr><td>Hyperreflexia (yes/no)</td><td>23/9 (71.9 / 28.1%)</td><td>13/11 (54.2 / 45.8%)</td><td>Chi<sup>2</sup> = 1.87<br>P = 0.17</td></tr><tr><td>Riluzole (yes/no)</td><td>17/15 (53.1 / 46.9%)</td><td>17/7 (70.8 / 29.2%)</td><td>Chi<sup>2</sup> = 1.80<br>P = 0.18</td></tr></tbody></table>

## 2.4. Recordings and stimulations

EMG activity was recorded using single-use bipolar surface electrodes (sticky foam electrodes with solid gel, 2-cm apart; FIAB, Florence, Italy) placed on cleansed skin (exfoliating cream and alcohol). Recordings were obtained from the triceps brachii (TB), flexor carpi ulnaris (FCU), flexor carpi radialis (FCR), vastus lateralis (VL, lateral head of quadriceps femoris), tibialis anterior (TA), soleus and gastrocnemius medialis (GM). Recording sites are detailed in Table S3 (Supplementary materials). EMG signals were amplified and filtered (x 1000-5000; 0.1-1 kHz bandpass; D360 8-channel Patient Amplifier, Digitimer Ltd., Hertfordshire, UK), digitized (2-kHz sampling rate; Power 1401 controlled by Signal 6.03, CED, Cambridge, UK) and stored on a personal computer for off-line analysis.

Percutaneous electrical stimulations (1-ms duration rectangular pulse; DS7A, Digitimer Ltd., Hertfordshire, UK) were delivered to ulnar, median, radial and superficial radial nerves (SRN), as well as to the femoral nerve (FN), common peroneal nerve (CPN), posterior tibial nerve (PTN), GM and sural nerves, using bipolar (cathode proximal to the spinal cord) or monopolar configurations (cathode over the nerve). Detailed electrode placements and stimulation configurations for each nerve are provided in Supplementary Table S3. Optimal stimulation sites were verified by observing motor responses (and H-reflex for Exp. 2a-c) in the corresponding EMG recordings, complemented by clinical palpation of tendon responses. Specifically, for CPN stimulation, a lower activation threshold for TA than for peroneal muscles was ensured (Simonetta-Moreau et al., 1999). For GM nerve stimulation, the absence of current spread to the PTN was confirmed by verifying the lack of Hoffman (H)-reflex in the GM EMG. For purely cutaneous nerves, electrode placement was validated by eliciting paresthesia in the corresponding sensory territory. For mixed nerves, the intensity of conditioning stimuli was normalized to both the motor threshold (x MT; minimal intensity producing the direct motor M response in the target EMG) and to the perceptual threshold (x PT; minimal intensity producing sensation). For SRN and sural nerve stimulation, the intensity was normalized to PT.

TMS was delivered using a 9-cm diameter round coil positioned over the optimal cortical site to elicit a motor evoked potential (MEP) in the TB (Exp. 1a-c). To ensure consistent coil placement across trials, participants wore a cap with marked coil coordinates. TMS intensity was set to the active motor threshold (AMT), defined as the minimum intensity required to evoke a TB MEP in <50% trials during tonic elbow extension at 10% maximal voluntary contraction (MVC). The level of muscle contraction was continuously monitored with visual feedback displaying the mean TB EMG normalized to that recorded during MVC.

## 2.5. Experimental protocols

A series of experiments were conducted on the upper (Exp. 1) and lower limbs (Exp. 2), with some participants taking part in both (see Supplementary Table S2 for the patient group; controls are indicated below, for each experiment), to assess synaptic transmission within spinal circuities involving excitatory and inhibitory interneurons. Experimental paradigms are summarized in Table 2. Demographic data for each experimental group are provided in Supplementary Table S1. Supporting methodological details and validation studies for these paradigms are provided in (Pierrot-Deseilligny and Burke, 2012; Pierrot-Deseilligny and Burke, 2005).

During testing, participants were comfortably seated in a reclining armchair with head support. For upper limb experiments involving the TB, the arm was fully extended and the hand positioned supine to facilitate inhibition from the SRN (Nielsen and Pierrot-Deseilligny, 1991). Participants used the armrest of the chair as resistance to assist with elbow extension. For experiments targeting wrist muscles, the forearm rested on the armrest. For lower-limb experiments, the tested leg was secured in an adjustable support to maintain the hip semiflexed
<caption>**Table 2**</caption>

Summary of experimental paradigms. The first column indicates the spinal cord level. The second column presents the experimental paradigms. The third column (Conditioning) lists the conditioning stimulations, with stimulation site and intensity indicated in brackets. The fourth column (Test response) indicates the recorded response, recording conditions (rest or tonic contraction, with force level in brackets), and response normalization (to $H_{max}$ or $M_{max}$) when applicable. The fifth column indicates the interstimulus intervals (ISIs) between conditioning and test stimulations (not applicable for Exp. 2d). The last column (Outcome measure) indicates the metric used to quantify excitation or inhibition. Abbreviations: AMT, active motor threshold; AUC, area under the curve; CPN, common peroneal nerve; FN, femoral nerve; GM, gastrocnemius medialis; H-reflex, Hoffmann reflex; ISI, interstimulus interval; MEP, motor evoked potential; $M_{max}$, maximum direct motor response; MVC, maximal voluntary contraction; PTN, posterior tibial nerve; RMS, root mean square; SRN, superficial radial nerve; TB, triceps brachii; TMS, transcranial magnetic stimulation.

<table><thead><tr><th></th><th>Experiment</th><th>Conditioning</th><th>Test response</th><th>ISI</th><th>Outcome measure</th></tr></thead><tbody><tr><td rowspan="10">Cervical</td><td>Exp. 1a: Propriospinal excitation</td><td>Ulnar nerve (elbow level)<br>Intensity<br>(0.75 x MT)</td><td>TB MEP<br>Tonic contraction (10% MVC)<br>TMS intensity<br>(1.2 x AMT)</td><td>Ulnar – TMS<br>(0–7 ms)</td><td>MEP AUC<br>(% mean test MEP)</td></tr><tr><td>Exp. 1b:<br>Inhibitory feedback of PNs</td><td>Ulnar nerve (elbow level)<br>Intensity<br>(1, 1.5 x MT)</td><td></td><td></td><td></td></tr><tr><td>Exp. 1c:<br>Cutaneous feedback of PNs</td><td>SRN<br>(wrist level)<br>Intensity<br>(8 x PT)</td><td></td><td>SRN – TMS<br>(5–12 ms)</td><td></td></tr><tr><td>Exp. 1d: Segmental inhibitions</td><td>Radial nerve<br>(arm level)<br>Intensity<br>(0.9–0.95 x MT)</td><td>FCR H-reflex<br>Rest<br>50% H<sub>max</sub></td><td>Radial – Median<br>(–1, 0, 1, 10, 13, 15 ms)</td><td></td></tr><tr><td>Exp. 2ab: Propriospinal excitation</td><td>CPN<br>(2 x MT)</td><td>Quadriceps H-reflex<br>Rest<br>Tonic contraction<br>(20% MVC)<br>15–30% M<sub>max</sub></td><td>CPN – FN<br>(7–10, 13–18 ms)</td><td>H-reflex amplitude<br>(% mean test H-reflex)</td></tr><tr><td rowspan="4">Lumbar</td><td>Exp. 2c: Segmental inhibitions</td><td>GM nerve<br>(0.95 x MT)<br>Sural nerve<br>(4 x PT)</td><td>Soleus H-reflex<br>Rest<br>15–30% M<sub>max</sub></td><td>GM – PTN<br>(4, 6, 8 ms)<br>Sural – GM<br>(4, 6, 8 ms)</td><td></td></tr><tr><td>Exp. 2d: Cutaneous reflexes</td><td>Sural nerve<br>14 shocks 333 Hz<br>≤ 6 x PT</td><td>TA ongoing EMG<br>Tonic contraction<br>(10% MVC)</td><td>–</td><td>EMG RMS<br>(% change from baseline)</td></tr></tbody></table>

(~80°), the knee semiextended (~130°), and the ankle in semi-plantarflexion (~100°).

The first series of experiments (Exp. 1; Table 2; Supplementary Table S1) examined synaptic transmission through i) the cervical propriospinal system, involving excitatory propriospinal neurons and interneurons mediating feedback inhibition, and ii) segmental inhibitory interneurons coordinating wrist muscle synergies:

- **Exp. 1a: Group I excitation mediated by cervical propriospinal neurons** (controls #1–43). The test response was a TB MEP elicited by TMS at 1.2 × AMT. Conditioning stimuli were delivered to the ulnar nerve at 0.75 × MT. The effect of ulnar nerve stimulation on TB MEPs was assessed at interstimulus intervals (ISIs) of 0–7 ms (1-ms increments) during tonic contraction at 10% MVC. Test TMS alone (n = 20) and paired stimulations (n = 20) were randomly alternated (0.66 Hz) within each session. Each ISI was tested in a separate session.

- **Exp. 1b: Group I feedback inhibition of cervical propriospinal neurons** (controls #29–43). Test and conditioning stimulations were as in Exp. 1a. At the optimal ISI for ulnar-induced MEP facilitation, conditioning intensity was increased to 1.0 and 1.5 x MT. Each recording session consisted of test (n = 20) and paired stimulations (n = 20), randomly alternated (0.66 Hz). Each intensity was tested in a separate session.

- **Exp. 1c: Cutaneous feedback inhibition of cervical propriospinal neurons** (controls #1–28). Test responses were as described for Exp. 1a and 1b, with conditioning stimuli delivered to the SRN at 8 × PT. ISIs ranged from 5 to 12 ms (1-ms increments) during tonic contraction at 10% MVC. Test TMS alone (n = 20) and paired stimulations (n = 20) were randomly alternated (0.66 Hz) within each session. Each ISI was tested separately.

- **Exp. 1d: Group I pre- and post-synaptic inhibitions in forearm flexors** (D1 inhibition; controls #26, 30–43, 45, 47, 48, 53, 57, 58). The test response was an H-reflex evoked by median-nerve stimulation in FCR EMG. Because H-reflex in forearm flexors are typically small (<15% the maximal M response [$M_{max}$]) (Sangari et al., 2024), the test reflex was adjusted to ~50% its maximum, where it is most sensitive to conditioning (Day et al., 1984). Conditioning stimuli were applied to radial nerve at 0.9–0.95 x MT, with ISIs ranging from –1 to +1 ms (short) and 10, 13 and 15 ms (long). Recordings were performed at rest; each acquisition consisted of test (n = 20) and paired stimulations (n = 20 × 3, for each ISI), randomly alternated (0.33 Hz). Separate sessions assessed short- and long-ISI effects.

The second series of experiments (Exp. 2; Table 2; Supplementary Table S1) investigated lumbar spinal circuits, including group I and II propriospinal neurons, non-reciprocal group Ib interneurons, and cutaneomuscular pathways:

- **Exp. 2ab: Group I and Group II excitations mediated by lumbar propriospinal neurons at rest and during tonic contraction** (controls #2–7, 9–25, 27, 28, 44). The test response was an H-reflex evoked by FN stimulation in VL EMG (15–30% $M_{max}$) (Crone et al., 1990). Conditioning stimuli were applied to CPN at 2 x MT, with ISIs of 7–10 ms (short) and 13–18 ms (long) at rest (Exp. 2a). During tonic knee extension at 20% MVC, FN stimulation was reduced to match $M_{max}$ range, and conditioning CPN stimuli were applied at the optimal ISIs identified at rest (Exp. 2b). Test and paired stimulations (n = 20 each) were randomly alternated (0.33 Hz).

- **Exp. 2c: Group Ib inhibition between ankle plantarflexors** (controls #30–32, 34–37, 39–42, 45–56). The test response was an H-reflex evoked by PTN stimulation in soleus EMG (15–30% $M_{max}$) (Crone et al., 1990). Conditioning stimuli were applied to the GM nerve at
0.95 x MT (ISIs 4, 6, 8 ms). At the optimal ISI for GM-induced inhibition, GM was combined with sural nerve stimulation (4 x PT) at 4, 6, and 8-ms ISIs. Recordings were performed at rest, with test (n = 20) and paired/triple stimulations (n = 20 × 3 per ISI) randomly alternated (0.33 Hz). One session used GM conditioning alone and two sessions, combined GM + sural inputs, to assess their interaction on the soleus H-reflex.

- **Exp. 2d: Cutaneomuscular responses in ankle dorsiflexors** (controls #29–37, 39–43, 45–48, 57). The effects of sural nerve stimulation on ongoing TA EMG were evaluated during tonic dorsiflexion (10% MVC). Conditioning trains (14 shocks, 333 Hz, ≤ 6 x PT) were delivered below nociceptive threshold and withdrawal reflex, but strong enough to elicit cutaneomuscular (Gibbs et al., 1995) and long-lasting responses in TA EMG (LLR, > 500 ms) (D'Amico et al., 2013). Each session included 50 stimulations (1 Hz); four sessions were conducted per participant (n = 4 × 50).

## 2.6. Quantification of neurophysiological metrics

The outcome measures for each paradigm are summarized in Table 2. TB MEPs were full-wave rectified, and the area under the curve (AUC) was calculated to quantify MEP size (Spampinato et al., 2023). AUC of test MEP was normalized to the pre-stimulus background EMG AUC, computed within a window of similar duration to the MEP itself. The effect of conditioning stimuli was expressed as the relative difference between conditioned and mean test MEPs, normalized to the mean test MEP.

$M_{max}$ and H-reflex amplitudes were measured peak-to-peak. The test H-reflex amplitude was normalized to $M_{max}$, and the effect of conditioning was calculated as the relative difference between conditioned and mean test H-reflex amplitudes, normalized to the mean test H-reflex.

For sural nerve stimulation, changes in ongoing TA EMG activity were quantified using the root mean square (RMS) value within analysis windows corresponding to successive facilitation and inhibition phases (E1, I1, E2, I2) (Gibbs et al., 1995). The relative modulation was obtained by normalizing the difference between post-stimulus and pre-stimulus RMS values to the mean pre-stimulus RMS.

## 2.7. Statistical analysis

All statistical analyses were performed using JMP® Pro 17 (SAS Institute Inc., Cary, NC, USA), with significance set at *P* < 0.05. For comparisons between ALS patients and controls, data normality was assessed using the Shapiro-Wilk test, and homogeneity of variances using Levene's test. Accordingly, group comparisons were performed using either Student's *t*-test or the Kolmogorov-Smirnov test.

Not all participants completed all experimental paradigms, resulting in an unbalanced dataset. Repeated-measures linear mixed models (RMLM) were therefore used, as they accommodate missing observations under the missing-at-random assumption and avoid listwise deletion. Missing data were thus not imputed. RMLM fitted by the least squares method were used to evaluate the effects of experimental and group-related factors, particularly when multiple conditions or repeated observations were present. When a significant main effect or interaction was identified, post hoc comparisons were performed using *t*-tests on least-squares means. To aid interpretation, the specific tests and within- or between-subject factors included in each model are described in the corresponding *Results* sections.

Associations between neurophysiological measures and clinical variables were analyzed using correlation or ANOVA, depending on the variable type. These analyses were limited to continuous neurophysiological metrics that showed significant group-level differences between ALS and control groups. In parallel, each individual ALS response was categorized as decreased, increased, or unaltered, relative to the 95% confidence interval (CI$_{95}$) of the control group. This categorical classification was applied to all tested metrics, regardless of group-level

significance, and subsequently used in a multiple correspondence analysis (MCA) to explore multivariate relationships between neurophysiological status and clinical parameters (functional impairment, disease stage, circuit type, and afferent origin), without imputation. The contribution of each variable to MCA dimensions was quantified using inertia (λ), and the statistical significance of the dimensions was assessed with $Chi^2$ tests.

Effect sizes were reported as Cramér's V for $Chi^2$ tests (Cramér, 2016), Cohen's *d* for pairwise *t*-tests and two-level model effects (Cohen, 1988; Rosnow and Rosenthal, 1996), and Cohen's *f*² for RMLM effects involving multiple levels or interactions (Selya et al., 2012). Effect size magnitudes were interpreted according to conventional benchmarks: Cohen's *d* (0.2 small, 0.5 medium, 0.8 large), Cohen's *f*² (0.02 small, 0.15 medium, 0.35 large), and Cramér's *V* (0.1 small, 0.3 medium, 0.5 large), following standard recommendations. All tests were two-tailed, and detailed statistical outputs (test statistics, degrees of freedom, *P* values, and effect sizes) are reported in the *Results* section.

Taken together, these methods allowed the systematic assessment of multiple spinal interneuron pathways, enabling direct comparison of their functional organization across disease stages and spinal levels.

# 3. Results

## 3.1. Group I excitation mediated by cervical propriospinal neurons to upper limb motoneurons

Excitation mediated by cervical propriospinal neurons to upper limb LMNs (Exp. 1a, Supplementary Table S1) was assessed by testing the effect of ulnar nerve stimulation on TB MEP during tonic voluntary contraction (Fig. 1A). As previously established, group I afferents from forearm flexors innervated by ulnar nerve do no project directly onto TB LMNs (Cavallari and Katz, 1989). Instead, they activate cervical propriospinal neurons, which also receive strong corticospinal inputs (Gracies et al., 1994; Gracies et al., 1991; Marchand-Pauvert et al., 2001). Ulnar-induced modulation of TB MEPs (Fig. 1B) was examined across several ISIs between ulnar nerve stimulation and TMS over the primary motor cortex. MEP facilitation occurred at ISIs between 0 and 5 ms (Fig. 1C), consistent with known conduction times of peripheral and corticospinal volleys and their convergence at the spinal level (Pierrot-Deseilligny, 1996).

To ensure reliable group comparisons, electrophysiological parameters that might influence the assessment of MEP facilitation (MVC and test MEP in TB, AMT to TMS, MT to ulnar nerve stimulation, ISIs) were first compared between groups; results are summarized in Table S4 (Supplementary materials). Only the test MEP and AMT to TMS differed significantly between ALS and control participants.

To account for the potential influence of test MEP on conditioned ones (Spampinato et al., 2023), cumulative ulnar-induced propriospinal facilitation of TB MEP across consecutive ISIs (e.g., 1–5 ms, Fig. 1C) was analyzed using a RMLM including Group (ALS vs. controls), Test MEP, and their interaction (Group x Test MEP). In the full dataset (adjusted $R^2$ = 0.52), no main effect reached statistical significance, although the estimated marginal means suggested greater MEP facilitation in ALS (Fig. S1AB, Supplementary materials).

Because not all participants were tested on their dominant side, particularly in the ALS group (Supplementary Table S1), and given the known asymmetry of propriospinal transmission with respect to handedness (Marchand-Pauvert et al., 1999a), a second analysis including only participants tested on their dominant side was performed. In this restricted dataset, ALS patients showed significantly greater ulnar-induced MEP facilitation compared to controls (adjusted $R^2$ = 0.45; *F* [1,66.8] = 6.08, *P* < 0.02, *d* = 0.5; Fig. 1DE). Neither the effect of Test MEP (*F* [1,32.4] = 0.002, *P* = 0.97, $f^2$ = 0.04) nor the Group x Test MEP interaction (*F* [1,32.4] = 0.11, *P* = 0.71, $f^2$ = 0.04) was significant. In contrast, patients tested on their non-dominant side exhibited markedly reduced MEP facilitation relative to those tested on their dominant side
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Ulnar nerve intensity (x MT)

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**Fig. 1.** Ulnar-induced TB MEP facilitation and its reversal into inhibition. (A) Schematic diagram illustrating the projection of group I afferents from the ulnar nerve onto excitatory C3-C4 propriospinal neurons (PN) and inhibitory interneurons (INs), which mediate feedback inhibition to PNs. These neurons regulate the excitation conveyed to triceps brachii (TB) motoneurons (MN) located between the C7 and T1 segmental levels. MN, PN, and the feedback inhibitory interneurons receive strong corticospinal inputs during voluntary contraction and transcranial magnetic stimulation (TMS) of the primary motor cortex. Excitatory and inhibitory synapses are represented by open triangles and filled circles at the ends of lines, respectively. (B) Mean TB electromyogram (EMG) from one control participant when TMS was applied alone (n = 20; test MEP in red) and when combined with ulnar nerve stimulation (0.75 x motor threshold, MT; n = 20; conditioned MEP in blue). Vertical interrupted lines indicate the window of analysis. (C) Mean difference between conditioned MEPs and mean test MEP (% of mean test MEP) in one control (as in B; open circles and dashed line) and one patient with ALS (filled circles and solid line), plotted against the interval between ulnar nerve stimulation and TMS (ms). Error bars represent ±1 standard error (SE). (D) Box plots illustrating the cumulative MEP facilitation (% of mean test MEP) in controls (N = 42; open box and circles) and patients with ALS (N = 29; filled box and circles), all tested on their dominant side. The lower limit of the box represents the 25th percentile, the upper limit the 75th percentile, the solid line within the box the mean, and the cross the median. Whiskers extend to the minimum and maximum values, and points outside the whiskers represent outliers. (E) Marginal means of cumulative MEP facilitation in control (open column) and ALS groups (filled column). Error bars indicate the 95%-confidence interval (CI). (F) Box plot (legend as in D) illustrating the influence of ulnar nerve stimulation intensity (x motor threshold, MT) on maximum MEP facilitation in subgroups of controls (N = 14) and patients with ALS (N = 14). (G) Marginal means of maximum MEP facilitation at the three ulnar nerve intensities tested (x MT). Error bars as in E. (H) Mean changes in MEP facilitation at intensities ≥1.0 x MT in controls (N = 14; open columns) and ALS patients (N = 14; filled columns). Error bars as in C. \*P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(27.8 ± 3.9 vs. 77.5 ± 12.6% of mean test MEP; Kolmogorov-Smirnov, D = 0.62, P < 0.05, d = 0.9).

Comparable findings were observed when analyzing the maximum MEP facilitation (Supplementary Figs. S1CD). Overall, these results indicate that ulnar-induced facilitation of TB MEP was enhanced in the patient group, particularly on the dominant side, suggesting that group I excitation mediated by cervical propriospinal neurons is likely increased in ALS.

## 3.2. Group I inhibitory feedback of cervical propriospinal neurons

The phylogenetic evolution in primates and humans is characterized by a strengthening of inhibitory control over cervical propriospinal neurons, mediated by both peripheral and descending pathways (feed-back inhibitory interneurons; Fig. 1A) (Alstermark et al., 1999; Nicolas et al., 2001). Therefore, an increase in propriospinal excitation, as observed in Exp. 1a, could reflect reduced inhibitory feedback onto these interneurons. To test this possibility, ulnar-induced facilitation of TB MEP was examined across increasing intensities of ulnar nerve stimulation (Nicolas et al., 2001). As in Exp. 1a, no differences in electrophysiological parameters (MVC and test MEP in TB, AMT to TMS, MT to ulnar nerve stimulation, ISIs) were found between groups except for test MEP size and AMT (Supplementary Table S4).

An RMLM (adjusted $R^2 = -0.17$) was fitted using Group (ALS vs. Controls), Test MEP, Intensity of ulnar nerve stimulation (0.75 vs. 1.0 vs. 1.5 x MT), along with all interactions. Although marginal means suggested greater MEP facilitation in ALS (ALS 27.3% the test MEP [$CI_{0.95} = 12.7-42.0$]) than in controls (14.4% [1.2-27.6]), the group effect did not reach statistical significance ($F[1,22.9] = 1.85, P = 0.19, d = 0.5; Fig. 1F$). Only the stimulation intensity significantly influenced MEP facilitation ($F[2,45.1] = 3.58, P < 0.05, f^2 = 0.02; Fig. 1G$). Post hoc pairwise comparisons revealed significant differences between 0.75 and 1.0 x MT ($t = -2.61, P < 0.02, d = 0.8$), and between 0.75 and 1.5 x MT ($t = -1.82, P_{unilateral} < 0.05, d = 0.6$), whereas the 1.0 and 1.5 x MT comparison was non-significant ($t = 0.78, P = 0.45, d = 0.1; Fig. 1G$).

Because test MEP had no significant influence and both groups displayed a similar suppression of MEP facilitation at intensities ≥1 x MT, a second model was used to test low vs. high-intensity only (0.75 vs. pooled 1.0–1.5 x MT). This model (adjusted $R^2 = 0.57$) again revealed a significant effect of stimulation intensity only ($F[1,26] = 14.18, P < 0.001, d = 0.8$). Finally, direct comparison of the change in MEP facilitation between 0.75 x MT and higher intensities confirmed that this decrement was similar across groups (t-test, $t = 0.12, P = 0.90, d = 0; Fig. 1H$).

Together, these results indicate that the reversal of ulnar-induced facilitation into inhibition was comparable between patients and control groups, suggesting that feedback inhibition of propriospinal neurons is likely preserved in ALS.

## 3.3. Cutaneous inhibitory feedback of cervical propriospinal neurons

The cervical propriospinal system receives convergent inputs from low-threshold muscular afferents (group I) and cutaneous afferents (Pierrot-Deseilligny, 1996). Although cutaneous afferents contribute less to propriospinal excitation than group I inputs (Burke et al., 1992a, 1992b; Gracies et al., 1991), they exert a strong inhibitory influence on descending transmission via feedback inhibitory interneurons (Fig. 2A) (Nielsen and Pierrot-Deseilligny, 1991). This inhibition is typically assessed by stimulating the SRN, a purely cutaneous branch.

SRN-induced suppression of TB MEP was examined under conditions similar to those used to assess group I inhibition (Exp. 1c; Fig. 2B), but with larger ISIs to account for the longer peripheral conduction time resulting from the more distal stimulation site at the wrist compared with the elbow (Fig. 2C). No differences in electrophysiological parameters were found between groups (MVC and test MEP in TB, AMT to TMS, PT to SRN stimulation, ISIs; Supplementary Table S4). Furthermore, no significant differences were observed between ALS patients and controls in either cumulative SRN-induced MEP depression (Kolmogorov-Smirnov, $D = 0.81, P = 0.53, d = 0.2; Fig. 2D$) or in its maximum amplitude (Student t-test, $t = 0.41, P = 0.69, d = 0.1$; Supplementary Fig. S1E). Similar results were obtained after excluding patients tested on their non-dominant side (Supplementary Table S1, Fig. S1EF).

Together, these findings indicate that cutaneous inhibition of propriospinal transmission was preserved in the patient group. Combined with the results of Exp. 1a and 1b, this supports the interpretation that the enhanced propriospinal transmission observed in ALS most likely reflects increased excitation mediated by cervical propriospinal neurons, rather than a reduction in their feedback inhibition (Figs. 1A and 2A).

## 3.4. Group I pre- and post-synaptic segmental inhibitions

Segmental pre- and post-synaptic inhibitions mediated by group I afferents are classically assessed using the D1 method (Pierrot-Deseilligny and Burke, 2012). To date, this approach has been applied only at the lumbar level in ALS patients (CPN-induced modulations of soleus H-reflex), where several studies, including one involving members of our team (Howells et al., 2020), have reported a significant reduction in presynaptic inhibition (Castro et al., 2024; Howells et al., 2020; Morin and Pierrot-Deseilligny, 1988; Sangari et al., 2016; Schieppati et al., 1985). Although our previous study primarily used threshold tracking to quantify conditioned H-reflexes, the classical amplitude-based method was also acquired but not published (Howells et al., 2020). After receiving this dataset, we extended the analysis by including French patients and age-matched controls, thereby addressing a major limitation of the original study (absence of age-matched controls). We confirmed a reduction in presynaptic inhibition and, additionally, we
<figure><img src="image_11.png" /><figcaption>**Fig. 2.** TB MEP depression by superficial radial nerve. (A) Schematic diagram illustrating the projection of cutaneous afferents from the superficial radial nerve (SRN) onto inhibitory interneurons (INs) mediating feedback inhibition to excitatory C3-C4 propriospinal neurons (PN). These neurons regulate the excitation conveyed to triceps brachii (TB) motoneurons (MN) located between the C7 and T1 segmental levels. MN, PN, and the feedback inhibitory interneurons receive strong corticospinal inputs during voluntary contraction and transcranial magnetic stimulation (TMS) of the primary motor cortex. Excitatory and inhibitory synapses as in Fig. 1. (B) Mean TB electromyogram (EMG) from one control participant (same as in Fig. 1B) when TMS was applied alone (n = 20; test MEP in red) and when combined with SRN stimulation (8 x perceptual threshold, PT; n = 20; conditioned MEP in black). Vertical interrupted lines indicate the window of analysis. (C) Mean difference between conditioned MEPs and mean test MEP (% of mean test MEP) in one control (as in B; open circles and dashed line) and one patient with ALS (filled circles and solid line; same as in Fig. 1C), plotted against the interval between SRN stimulation and TMS (ms). Error bars represent ±1 SE. (D) Box plots (as in Fig. 1D) illustrating the cumulative MEP inhibition (% of mean test MEP) in controls (N = 28; open box and circles) and patients with ALS (N = 19; filled box and circles). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

found a significant reduction in post-synaptic inhibition (Fig. S2, Supplementary materials).

To determine whether similar alterations occur at the cervical level, we examined the effect of radial nerve stimulation on FCR H-reflex in both controls and patients (Fig. 3A; Exp. 1d, Supplementary Table S1). Short ISIs (–1, 0, +1 ms) were used to assess segmental post-synaptic inhibition (Fig. 3B–D), whereas longer ISIs (10, 13, 15 ms) were used to evaluate presynaptic inhibition (Fig. 3E–G). No differences in electrophysiological parameters (Mmax and test H-reflex in FCR, MT to radial nerve stimulation, ISIs) were found between groups except for the H-reflex test size (Supplementary Table S4).

RMLMs were used to compare ALS patients and controls, accounting for the influence of the test H-reflex on conditioned responses (Crone et al., 1990). For cumulative post-synaptic inhibition, the model revealed no significant effect (adjusted R² = -0.07): i) Group (ALS vs.

Controls): F(1,39) = 0.39, P = 0.53, d = 0.3, ii) Test H-reflex: F(1,39) = 0.27, P = 0.61, d = 0.7, iii) Group x Test H-reflex: F(1,39) = 0.54, P = 0.47, f² = 0.01. In contrast, the model assessing cumulative presynaptic inhibition revealed a significant reduction in ALS (R² = 0.09; F[1,38.9] = 4.13, P < 0.05, d = 0.8). Neither the test H-reflex (F[1,5.7] = 0.59, P = 0.47, d = 0.7) nor the Group x Test H-reflex interaction was significant (F[1,5.7] = 1.08, P = 0.34, f² = 0). Similar results were obtained when comparing the maximum inhibition (Fig. S3, Supplementary materials).

All these results indicate that cervical group I post-synaptic inhibition was preserved in patients but not at the lumbar level, whereas presynaptic inhibition of group Ia afferents was significantly reduced at both levels.
<figure><img src="image_12.png" /><figcaption>**Fig. 3.** FCR H-reflex reduction by radial nerve stimulation. (A) Schematic diagram illustrating the projection of group I afferents from the radial nerve (blue) onto group I inhibitory interneurons (INs) mediating post-synaptic inhibition to flexor carpi radialis (FCR) motoneurons (MN) and onto primary afferent depolarization (PAD) INs mediating presynaptic inhibition of group Ia terminals from FCR (red) activated by median nerve stimulation (eliciting H-reflex in FCR electromyogram [EMG]: group Ia-MN loop, shown in red)). (B,E) Mean FCR EMG from one control participant when median nerve was applied alone (n = 20; test H-reflex in red) and when combined with radial nerve stimulation (0.9 x motor threshold, MT; n = 20; conditioned H-reflex in blue) with 1 (B) or 13 ms interstimulus interval (ISI) (E). Vertical interrupted lines indicate the window of analysis. (C,F) Mean difference between conditioned H-reflex and mean test H-reflex (% of mean test H-reflex) in one control (open circles and dashed line) and one patient with ALS (filled circles and solid line), plotted against the interval between radial and median nerve stimulations (ms; same participants in C and F). Error bars represent ±1 SE. (D,G) Box plots (as in Fig. 1D) illustrating the cumulative H-reflex inhibition (% of mean test H-reflex) in controls (N = 21; open box and circles) and patients with ALS (N = 22; filled box and circles) at short ISIs for post-synaptic inhibition (D) and at longer ISIs for presynaptic inhibition (G). \*P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
### 3.5. Group I-II excitations mediated by lumbar propriospinal neurons to lower limb motoneurons

Group I afferents from TA do no project directly onto quadriceps LMNs (Meunier et al., 1993). Instead, together with group II afferents, they project onto lumbar propriospinal neurons (Forget et al., 1989; Simonetta-Moreau et al., 1999), which also receive strong corticospinal inputs (Fig. 4A) (Forget et al., 1989; Marchand-Pauvert et al., 1999b). To evaluate propriospinal transmission at the lumber level, CPN-induced changes in quadriceps H-reflex (Fig. 4B) were assessed at short (7–11 ms) and long ISIs (13–18 ms), which were optimal for group I- and group II-mediated propriospinal excitation, respectively (Exp. 2ab, Supplementary Table S1; Fig. 4C) (Marque et al., 1996).

After confirming the absence of group differences in electrophysiological parameters (MVC, $M_{max}$ and test H-reflex in quadriceps, MT to CPN stimulation, ISIs; Supplementary Table S4), an RMLM was applied to assess the factors influencing CPN-induced facilitation of the quadriceps H-reflex. The model included Group (ALS vs. Controls), State (rest vs. contraction), ISI (short vs. long ISIs), and their interactions. Fig. 4D illustrates the distribution of data across groups and conditions. The model (adjusted $R^2 = 0.74$) identified a significant increase in H-reflex facilitation in patients compared with controls ($F[1,45] = 8.20, P < 0.01, d = 0.5; Fig. 4E$). Additionally, H-reflex facilitation was significantly reduced during tonic contraction relative to the resting state ($F[1,45] = 30.78, P < 0.001, d = 0.6; Fig. 4F$). A significant State × ISI interaction was detected ($F[1,45] = 10.79, P < 0.01, f^2 = 0.5$), and post hoc pairwise comparisons showed a substantial decline in facilitation during contraction at both ISIs (short ISIs: $t = -2.16, P < 0.05, d = 0.4$; long ISIs: $t = -6.36, P < 0.001, d = 0.9; Fig. 4G$). No significant differences were found between short and long ISIs ($F[1,45] = 0.55, P = 0.46, d = 0.1$). Moreover, the Group x State ($F[1,45] = 1.18, P = 0.28, f^2 = 0.2$) and Group x ISI ($F[1,45] = 0.01, P = 0.93, f^2 = 0.2$) interactions were not statistically significant. Finally, the mean reduction in facilitation from rest to contraction, pooled across short and long ISIs, did not differ between groups (t-test, $t = 1.07, P = 0.29, d = 0.3; Fig. 4H$).

Overall, these findings indicate that CPN-induced quadriceps H-reflex facilitation was enhanced in patients compared with controls across all ISIs, whereas in both groups the facilitation was similarly depressed during tonic contraction. These results suggest that excitation mediated by both group I and group II pathways involving lumbar propriospinal circuits is likely strengthened in patients with ALS.

We have previously shown that the depression of propriospinal excitation during contraction is mediated by non-reciprocal group I inhibition, involving Ib interneurons acting at the motoneuron level and truncating the H-reflex; activation of these interneurons results from the convergence of peripheral and descending inputs, particularly evident when using this experimental paradigm (Fig. 4A) (Iglesias et al., 2008; Marchand-Pauvert et al., 2002). These findings therefore suggest that Ib inhibition may be preserved in ALS.

### 3.6. Autogenetic group Ib inhibition in ankle plantarflexors

Inhibition through Ib interneurons was further examined using a well-established paradigm assessing the soleus H-reflex following stimulation of the GM nerve branch (Fig. 5A), either alone (Fig. 5B) or in combination with sural nerve stimulation (Fig. 5A,C; Exp. 2c, Supplementary Table S1), which enhances Ib inhibition (Pierrot-Deseilligny et al., 1981; Pierrot-Deseilligny et al., 1979). GM-induced H-reflex reduction was observed at ISIs 4-8 ms (Fig. 5D), and a 4-to-8-ms shift in combined condition due to the extra peripheral conduction time for sural cutaneous inputs (Fig. 5E; Supplementary Table S3) (Pierrot-Deseilligny et al., 1981; Pierrot-Deseilligny and Burke, 2012). The maximum inhibition observed in each participant, whether from GM stimulation alone or from combined stimulation, was retained for group comparison.

No differences in electrophysiological parameters were found between groups ($M_{max}$ and test H-reflex in soleus, MT to GM nerve, PT to SRN stimulation, ISIs; Supplementary Table S4). The proportion of controls and patients showing maximum inhibition during combined sural and GM nerve stimulation was similar ($Chi^2 = 0.38, P = 0.54, V = 0.1$). Pairwise comparison revealed no significant group differences in the magnitude of maximum inhibition (ALS $-9.5 \pm 2.9$ vs. Controls $-14.6 \pm 3.0\%$ of the mean test H-reflex; Kolmogorov-Smirnov, $D = 0.32, P = 0.18, d = 0.4; Fig. 5F$).

These findings, consistent with those from Exp. 2ab involving the quadriceps H-reflex, further indicate that group Ib inhibition was not significantly altered in the ALS group.

### 3.7. Cutaneomuscular reflexes in ankle dorsiflexors

The cutaneous influence on spinal cord circuitry was further examined by assessing the effect of sural nerve stimulation on TA EMG (Fig. 6A) (D'Amico et al., 2013; Gibbs et al., 1995). This approach is commonly used to evaluate cutaneomuscular reflexes, which appear in averaged rectified EMG as successive phases of increased and decreased activity. To focus the analysis on spinal mechanisms, only the initial phases of excitation and inhibition, referred to as E1 and I1 (Fig. 6 BC), were analyzed, as later responses are contaminated by transcortical loop (Gibbs et al., 1995; Nielsen et al., 1997; Tarkka, 1986) (Fig. 6A). We also examined whether long-lasting EMG bursts were present during the long-lasting reflex (LLR) (Fig. 6D) (D'Amico et al., 2013).

Among electrophysiological parameters (TA MVC, PT to sural nerve stimulation, and E1/I1 latencies and durations), PT to single-shock sural nerve stimulation was significantly higher in patients (Supplementary Table S4). The conditioning stimulation train, normalized to PT, was progressively increased up to 6 x PT. However, this intensity was painful in four patients and five controls, and it triggered a withdrawal reflex in four additional patients ($\ge 4.5$ x PT). All four of these patients exhibited hyperreflexia, and only one showed spasticity (patients #30, 31, 47, 48; Supplementary Table S2), similar to others who did not show withdrawal reflex even at 6 x PT. No control exhibited a withdrawal reflex, and no significant group difference was detected ($Chi^2 = 4.47, P < 0.05, V = 0.34; Fig. 6E$). Consequently, the conditioning train intensity was adjusted to just below the threshold for pain and withdrawal reflex, and no group differences were found in stimulation intensity (Supplementary Table S4).

Eliciting E1 in TA EMG can be challenging in healthy individuals, as reported previously (Gibbs et al., 1995; Nielsen et al., 1997; Tarkka, 1986). Consistent with this, we observed a greater number of controls lacking an E1 response (6 out of 19) compared to patients (3 out of 19) although this difference was not statistically significant ($Chi^2 = 1.31, P = 0.25, V = 0.19; Fig. 6F$). A RMLM (adjusted $R^2 = 0.33$) was applied with Group (ALS vs. Control; Fig. 6GH), Sural nerve PT, and Response type (E1 vs. I1) as predictors, including their interactions. Neither Group nor Sural nerve PT significantly contributed to the model ($F_{group}[1,2.3] = 1.42, P = 0.34, f^2 = 0.14; F_{PT}[1,1.0] = 0.01, P = 0.93, f^2 = 0.13$). As expected, the difference between responses (E1 vs. I1) was significant ($F[1,20.5] = 24.47, P < 0.001, d = 0.3$), but no significant Group x Response interaction was detected ($F[1,20.5] = 0.20, P = 0.66, f^2 = 0.18$). These findings indicate that E1 and I1 were comparable between ALS patients and controls (Fig. 6I).

Although no statistical outliers were detected (Fig. 6GH), two patients exhibited markedly large E1 responses relative to the rest of the group (Fig. 6G; patients# 24, 47, Supplementary Table S3). Notably, both were untreated with riluzole treatment, and one exhibited hyperreflexia, similar to other patients who did not show exaggerated responses. To evaluate their potential impact, the model was recalculated after excluding these two individuals. The results remained unchanged, and the group difference, particularly for E1, was even smaller (Fig. S4, Supplementary materials).

Lastly, we compared the time at which the post-stimulus EMG
<figure><img src="image_13.png" /><figcaption>**Fig. 4.** CPN-induced quadriceps H-reflex facilitation and its depression during tonic contraction. (A) Schematic diagram illustrating the projection of group I and group II afferents from the common peroneal nerve (CPN) onto excitatory T12-L1 propriospinal neurons (PN). These neurons regulate the excitation transmitted to quadriceps motoneurons (MN) located between the L2 and L4 segmental levels, assessed using the Hoffmann (H)-reflex in quadriceps (mediated by the group Ia-MN loop, shown in red) activated by femoral nerve (FN) stimulation. CPN afferents, including joint and cutaneous fibers, as well as group I afferents from the FN, converge onto group Ib interneurons (INs), which inhibit quadriceps MN. MN, PN, and group Ib interneurons receive strong corticospinal inputs during voluntary contraction. Excitatory and inhibitory synapses as in Fig. 1. (B) Mean vastus lateralis (VL) electromyogram (EMG) from one control participant when FN was applied alone (n = 20; test H-reflex in red) and when combined with CPN stimulation (2 x MT; n = 20; conditioned H-reflex in blue). Vertical interrupted lines indicate the window of analysis. (C) Mean difference between conditioned H-reflexes and mean test H-reflex (% of mean test H-reflex) in one control (as in B; open circles and dashed line) and one patient with ALS (filled circles and solid line), plotted against the interval between CPN and FN stimulations (ms). Error bars represent ±1 SE. (D) Box plots (as in Fig. 1D) illustrating the maximum H-reflex facilitation (% of mean test H-reflex) in controls (N = 26; open box and circles) and patients with ALS (N = 21; filled box and circles) across conditions (rest in light blue vs. tonic contraction in dark blue). (E-G) Marginal means of H-reflex facilitation estimated from the model in the group of controls (open column) and patients (filled column; E), in resting state (light blue dotted column) and during contraction (dark blue dotted column; F), and at short and long ISIs in both states (resting state in light blue vs. contraction in dark blue; G). Error bars indicate the 95%-CI. (H) Mean difference in H-reflex facilitation between resting state and tonic contraction in controls (open column) and patients with ALS (filled column). Error bars are ±1 SE. \*P < 0.05, \*\*P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**Fig. 5.** GM and sural nerve-induced soleus H-reflex inhibition. (A) Schematic diagram illustrating the projection of group Ib afferents from the gastrocnemius medialis (GM) nerve onto group Ib interneurons (INs), and their control by excitatory INs receiving cutaneous inputs from sural nerve. The Ib inhibition transmitted to soleus motoneurons (MN) was assessed using the H-reflex in soleus (group Ia-MN loop, shown in red), activated by posterior tibial nerve (PTN) stimulation. Excitatory and inhibitory synapses as in Fig. 1. (BC) Mean soleus electromyogram (EMG) from one control participant when PTN was applied alone (n = 20; test H-reflex in red) and when combined with GM stimulation (0.95 x MT; n = 20; conditioned H-reflex in blue; B), or when GM stimulation was preceded by sural nerve stimulation (4 x PT; n = 20; conditioned H-reflex in black; C). Vertical interrupted lines indicate the window of analysis. (DE) Mean difference between conditioned H-reflexes and mean test H-reflex (% of mean test H-reflex) in one control (as in B; open circles and dashed line) and one patient with ALS (filled circles and solid line), plotted against the interval between GM and PTN stimulations (ms; D) or sural nerve and GM stimulations (ms; the interval between GM and PTN being 8 and 6 ms in the control and in the patient, respectively; E). Error bars represent ±1 SE. (F) Box plots (as in Fig. 1D) illustrating the maximum H-reflex inhibition (% of mean test H-reflex) in controls (N = 23; open box and circles) and patients with ALS (N = 25; filled box and circles). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

returned to baseline to determine whether any significant activity persisted beyond 500 ms, which would be compatible with an LLR. In none of the participants did post-stimulus EMG changes extend beyond 500 ms, and no significant difference was found between groups (ALS 310.2 ± 22.9 vs. Controls 310.3 ± 17.6 ms; Student t-test, t = 0.003, P = 0.99, d = 0).

Together, these results indicate that cutaneomuscular reflex transmission at the ankle level, including E1 and I1 components and LLR, was not significantly modified in patients, suggesting that synaptic activity at this level is likely preserved in ALS.

## 3.8. Connections between spinal circuit transmission and key clinical features

At the group level, propriospinal excitation and presynaptic inhibition of group Ia terminals were significantly altered in patients compared with controls. Correlation analyses were conducted to

evaluate potential associations between the levels of excitation or inhibition and various clinical features, including site of onset, disease duration, ALSFRS-r score, clinical predominant form, spasticity, riluzole treatment, hyperreflexia, and mean progression rate (Supplementary Table S2). No significant associations were found, except for lumbar propriospinal excitation, which was significantly higher in patients with spasticity than in those without (Table 3).

To further examine the link with clinical status, patient results were classified as unaltered, increased, or decreased based on the CI95 of the control group for each neurophysiological measure. MCA was performed to test whether ALS-related status (unaltered vs. increased vs. decreased) was influenced by the type of spinal circuitry tested (excitation vs. inhibition), the stage of the disease (early vs. late), and the degree of functional impairment (weak vs. moderate vs. strong). All spinal circuits tested were categorized not only by the valence of the connection (excitatory vs. inhibitory), but also by whether their main afferent input originated from muscle spindles (group Ia-II afferents;
<figure><img src="image_15.png" /><figcaption>**Fig. 6.** Sural nerve induced TA EMG modulations. (A) Schematic diagram showing the projection of cutaneous afferents from the sural nerve (dotted black line) onto interneurons mediating the cutaneomuscular responses to tibialis anterior (TA) motoneurons (MN), as assessed by electromyogram (EMG) recorded during tonic ankle dorsiflexion. TA MNs and interneurons mediating cutaneous inputs receive corticospinal inputs (red line). Excitatory and inhibitory synapses as in Fig. 1. (BC) Mean rectified TA EMG (n = 200) from one control participant (B: intensity of sural nerve = 6 x PT) and one patient with ALS (C; 6 x PT) around stimulation train (14 pulses, 333 Hz) applied to the sural nerve. Vertical interrupted lines indicate the window of analysis for the first phases of EMG facilitation (E1) and depression (I1). (D) Mean TA EMG (n = 200) around stimulation train to sural nerve (6 x PT, 14 pulses, 333 Hz) from another patient; first burst after the train corresponds to the long-latency polysynaptic reflex (LPR, including E1 and I1) and the late burst, to the long-lasting reflex (LLR) (D'Amico et al., 2013). (EF) Proportion of participants in both groups (Controls vs. ALS) with (grey bars) or without (open bars) withdrawal reflex due to sural nerve stimulation train (intensity ≥4.5 x PT; E), and with (grey bars) or without (open bars) the first phase of EMG facilitation, E1 (F). (FG) Box plots (as in Fig. 1D) illustrating the extra root mean square (RMS) (% of pre-stimulus RMS) after sural nerve stimulation during E1 (F) and I1 (G) in controls (N = 19; open box and circles) and patients with ALS (N = 19; filled box and circles). (H) Marginal means of E1 (left part) and I1 (right part) in control (open circles and interrupted line) and ALS groups (filled circles and continuous line). Error bars indicate the 95%-confidence interval. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

propriospinal excitations, presynaptic inhibition of group Ia terminals, reciprocal group Ia inhibition at lumbar level) or from other sources (propriospinal inhibitions, group Ib inhibitions including post-synaptic group I inhibition at cervical level [non-reciprocal in origin; (Pierrot-Deseilligny and Burke, 2012)], cutaneomuscular responses).

The MCA showed that the first two dimensions together explained the full variance in the dataset, with Dimension 1 accounting for 76.2% and Dimension 2 for 23.8% (Fig. 7A). Both axes were statistically significant (Dimension 1: $Chi^2 = 65.95, P < 0.001, V = 0.18$; Dimension 2: $Chi^2 = 20.59, P < 0.01, V = 0.10$), indicating a structured relationship between the ALS-related neurophysiological status (decreased,

increased, or unaltered) and the tested factors. Category contributions to each dimension sum to 100%. The average expected contribution was therefore defined as 100 divided by the number of categories (N = 12), and categories exceeding 8.3% were considered as the main contributors structuring the dimensions (Greenacre, 2017). Dimension 1 primarily captured the contrast between the changes in spinal circuit activity (decreased vs. increased vs. unaltered; Fig. 7B). Variables contributing most strongly to this dimension included excitatory responses and inhibition principally mediated by muscle spindle afferents. In contrast, Dimension 2 separated increased from unaltered responses and was mainly driven by excitatory circuits, particularly those mediated by
<caption>**Table 3**</caption>

Links between altered excitations and inhibitions in patient group with main clinical features. The first column lists clinical features (see Supplementary Table S3). The next three columns present the statistical analysis results for each neurophysiological measure significantly modified in the sub-groups of patients, compared to controls: cervical propriospinal excitation, presynaptic inhibition of group Ia terminals (cervical level), and lumbar propriospinal excitation. In each cell: first line: type of statistical test used, second line: test statistic with degrees of freedom or 95% confidence interval, third line: *P* value (with * indicating statistical significance, *P* < 0.05).

<table><thead><tr><th>Clinical features</th><th>Cervical propriospinal excitation</th><th>Presynaptic inhibition of group Ia terminals</th><th>Lumbar propriospinal excitation</th></tr></thead><tbody><tr><td>Onset site</td><td>ANOVA<br>F(2,26) = 0.50<br><i>P</i> = 0.61</td><td>ANOVA<br>F(2,19) = 0.47<br><i>P</i> = 0.63</td><td>ANOVA<br>F(2,18) = 0.51<br><i>P</i> = 0.61</td></tr><tr><td>Disease duration</td><td>Correlation analysis<br><i>r</i> = -0.02 (-0.39, 0.35)<br><i>P</i> = 0.91</td><td>Correlation analysis<br><i>r</i> = 0.17 (-0.27, 0.55)<br><i>P</i> = 0.45</td><td>Correlation analysis<br><i>r</i> = -0.08 (-0.49, 0.37)<br><i>P</i> = 0.74</td></tr><tr><td>ALSFRS-r</td><td>Correlation analysis<br><i>r</i> = -0.01 (-0.37, 0.36)<br><i>P</i> = 0.97</td><td>Correlation analysis<br><i>r</i> = -0.09 (-0.50, 0.34)<br><i>P</i> = 0.67</td><td>Correlation analysis<br><i>r</i> = 0.07 (-0.37, 0.49)<br><i>P</i> = 0.75</td></tr><tr><td>Form</td><td>ANOVA<br>F(2,26) = 0.30<br><i>P</i> = 0.74</td><td>ANOVA<br>F(2,19) = 0.61<br><i>P</i> = 0.55</td><td>ANOVA<br>F(2,18) = 1.25<br><i>P</i> = 0.31</td></tr><tr><td>Spasticity</td><td>ANOVA<br>F(1,27) = 0.03<br><i>P</i> = 0.86</td><td>ANOVA<br>F(1,20) = 0.94<br><i>P</i> = 0.34</td><td>ANOVA<br>F(1,19) = 6.13<br><i>P</i> < 0.05*</td></tr><tr><td>Riluzol</td><td>ANOVA<br>F(1,27) = 0.29<br><i>P</i> = 0.59</td><td>ANOVA<br>F(1,20) = 2.43<br><i>P</i> = 0.13</td><td>ANOVA<br>F(1,19) = 0.19<br><i>P</i> = 0.67</td></tr><tr><td>Hyperreflexia</td><td>ANOVA<br>F(1,27) = 0.13<br><i>P</i> = 0.72</td><td>ANOVA<br>F(1,20) = 1.06<br><i>P</i> = 0.31</td><td>ANOVA<br>F(1,19) = 0.61<br><i>P</i> = 0.45</td></tr><tr><td>Mean progression rate</td><td>Correlation analysis<br><i>r</i> = -0.07 (-0.43, 0.30)<br><i>P</i> = 0.71</td><td>Correlation analysis<br><i>r</i> = -0.14 (-0.53, 0.30)<br><i>P</i> = 0.54</td><td>Correlation analysis<br><i>r</i> = -0.12 (-0.52, 0.33)<br><i>P</i> = 0.61</td></tr></tbody></table>

cutaneous afferents (E1; Fig. 7C), suggesting that this axis captured a qualitative distinction between preserved and altered response patterns. These dimensions were then used to examine how specific circuit types and clinical factors (functional impairment and disease stage) relate to patterns of spinal circuit dysfunction.

To test whether spinal circuits primarily driven by muscle spindle afferents are more affected in ALS than other circuits, we compared the distribution of neurophysiological statuses across the four circuit categories. Within excitatory circuits, a significant difference was found between those primarily mediated by muscle spindle afferents and those receiving inputs from other sources (*Chi²* = 7.41, *P* < 0.05, *V* = 0.29), with the former more frequently showing increased responses, particularly in patients with severe functional impairment (Fig. 7D). In contrast, no significant difference was observed among inhibitory circuits (*Chi²* = 1.84, *P* = 0.40, *V* = 0.11), suggesting a generally uniform depression across inhibitory pathways. However, qualitative inspection of the data (Fig. 7DE) suggests that inhibitory circuits mediated by muscle spindle afferents may be more frequently altered than those receiving input from other sources. These findings support the view that excitatory pathways primarily mediated by muscle spindle afferents undergo selective, stage-dependent modulation, whereas inhibitory dysfunction appears earlier and may also preferentially affect spindle-driven circuits.

To further investigate how circuit alterations evolve with disease severity, we examined the distribution of neurophysiological responses across categories of functional impairment (Weak, Moderate, Severe). When restricting the analysis to excitatory circuits, a significant

association was observed between response type and clinical severity (*Chi²* = 7.41, *P* < 0.05, *V* = 0.29), with increased excitatory effects being more frequent in severely impaired patients. This pattern indicates a progressive enhancement of excitation as the disease advances. In contrast, no significant differences were found for inhibitory circuits across the same clinical categories (*Chi²* = 1.84, *P* = 0.40, *V* = 0.11), consistent with the notion that inhibitory dysfunction arises early and remains uniformly altered throughout the disease course.

## 4. Discussion

Among the spinal excitatory pathways tested, only those conveyed by cervical and lumbar propriospinal interneurons were significantly enhanced in the group of patients, whereas cutaneomuscular excitation (E1) remained comparable to controls. Presynaptic inhibition of group Ia terminals was reduced at both spinal levels, while most tested postsynaptic inhibitory mechanisms appeared preserved. Notably, only reciprocal group Ia inhibition at the lumbar level was significantly reduced in ALS patients. When comparing patients and controls as whole groups, these findings may suggest that non-monosynaptic excitatory spinal transmission is more affected than inhibitory control, and that interneuron circuits driven by group Ia and group II afferents are particularly vulnerable compared to those receiving group Ib or cutaneous inputs. Analyses incorporating clinical status further confirmed this afferent-driven distinction and also revealed that the apparent imbalance between excitatory and inhibitory impairments reflects a stage-dependent pattern: increased excitatory responses were more frequent in patients with the most severe functional impairment, while inhibitory alterations, whether pre- or postsynaptic, were already present at early stages, when functional impairment was still mild. Taken together, these results suggest that spinal circuit dysfunction in patients with ALS follows a temporally organized and afferent-driven trajectory, in which early-onset failure of inhibition, particularly in circuits mediated by muscle spindle afferents, is followed by a progressive amplification of excitation as the disease progresses (Fig. 8).

### 4.1. Spinal circuits and interneurons: organization and function

Several decades of research have led to the development and validation of the methods employed here to describe the organization of spinal microcircuits in humans (Pierrot-Deseilligny and Burke, 2012; Pierrot-Deseilligny and Burke, 2005). These methods have also been extensively used to evaluate the impact of neurological lesions on synaptic activity within spinal networks, particularly after stroke, spinal cord injury, or in patients with Parkinson's disease or dystonia. The spinal neural networks investigated involve various types of interneurons that have been thoroughly characterized in animal models, primarily the cat, both anatomically and functionally, including their regulation by supraspinal centers and especially their main sensory inputs (Jankowska, 1992). This body of work has demonstrated a high degree of conservation in the organization of spinal networks across mammals. However, phylogenetic adaptations in connectivity have occurred in relation to bipedal locomotion and the resulting functional release of the upper limbs from gait-related roles in humans (Pierrot-Deseilligny and Burke, 2012; Pierrot-Deseilligny and Burke, 2005; Sengupta and Bagnall, 2023). Despite recent technological and methodological advances in spinal functional neuroimaging (Landelle et al., 2021), indirect electrophysiological techniques remain, to date, the only means for mechanistically probing spinal networks and interneurons in humans. The development of genetic tools and the generation of preclinical models of human diseases have, over the past three decades, prompted increased interest in spinal interneurons in the mouse model, including efforts to characterize them genetically in order to manipulate their expression (Alaynick et al., 2011). Although direct correspondence with the functionally defined interneurons described in classical spinal network models is not always straightforward, many of the same
<figure><img src="image_16.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_20.png" /></figure>

**Fig. 7.** Influence of disease stage and functional impairment on ALS-related changes in spinal excitatory and inhibitory circuits, depending on whether they are primarily mediated by muscle spindle afferents. (A) Biplot showing the projection of variable modalities on the two main multiple correspondence analysis (MCA) dimensions (X and Y axes), which reflect their inertia (λ; deviation from independence; higher values indicate stronger associations). Modalities include ALS-related changes in spinal circuitry (status according to the 95% confidence interval relative to controls; red circles), excitatory and inhibitory circuit types (diamonds), distinguished by their primary afferent source - from muscle spindles (blue symbols) or not (black symbols) -, disease stage (yellow circles), and functional impairment level (green circles). (BC) Bar plot showing the contribution of each category to Dimension 1 (B) and Dimension 2 (C) of the MCA; same legend as in A. The horizontal red line indicates the average expected contribution (8.3%), calculated as 100 divided by the number of categories included in the analysis (N = 12). Categories exceeding this threshold contribute most strongly to the construction of this dimension. (DE) Radar plots illustrating the proportion of patients with ALS showing decreased (blue), increased (red), or unaltered (grey) responses across excitatory and inhibitory spinal circuits receiving input primarily from muscle spindles (B) or from other sources (C). Each axis represents the proportion of patients (%) in a given functional impairment category (Weak, Moderate, Severe), allowing for a visual comparison of response patterns across different levels of disease severity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
<figure><img src="image_21.png" /><figcaption>**Fig. 8.** Schematic overview of spinal circuit alterations across disease stages in ALS. (A-C) Schematics summarizing the spinal circuits investigated in the present study and the inferred direction of changes in excitatory and inhibitory influences across disease progression. The left side of the spinal cord depicts circuits predominantly driven by muscle spindle afferent inputs, engaging propriospinal and segmental interneuronal networks. The right side illustrates circuits mainly driven by cutaneous afferents and Golgi tendon organs, involving cutaneomuscular and non-reciprocal inhibitory pathways. In healthy controls (A), spinal motor output reflects a balanced interaction between excitatory (E) and inhibitory (I) inputs, shaped by afferent drive. In early-stage ALS (B), a reduction in inhibitory influences combined with preserved or increased excitation leads to an altered excitation/inhibition (E/I) ratio, with changes depending on the nature of the afferent input. In advanced stages of the disease (C), inhibitory mechanisms are further reduced, while excitatory drive is increased, resulting in a marked imbalance of spinal network activity. Arrows indicate the direction and relative magnitude of changes in excitation and inhibition inferred from the experimental findings. (D) Conceptual model shows the changes in excitation (E; green line) and inhibition (I; black line) across disease progression, with early disinhibition followed by progressively enhanced excitation, leading to an increased E/I ratio over the course of the disease. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

interneuron populations identified in cats and humans have also been recognized in mice (Sengupta and Bagnall, 2023).

Short propriospinal interneurons, located above the motor columns at cervical and lumbar levels, have widespread projections onto motoneurons innervating the muscles of the upper and lower limbs (Chaix et al., 1997; Forget et al., 1989; Pierrot-Deseilligny, 1996; Pierrot-Deseilligny and Marchand-Pauvert, 2002; Simonetta-Moreau et al., 1999). These neurons mediate and regulate the descending motor command *en route* to motoneurons by integrating sensory inputs from various sources (Marchand-Pauvert et al., 1999b; Pauvert et al., 1998). However, excitations driven by group I and II muscle afferents are generally stronger than those elicited by cutaneous inputs, suggesting a predominant contribution from muscle spindle afferents (Forget et al., 1989; Malmgren and Pierrot-Deseilligny, 1988; Marque et al., 2005). The low threshold of group I responses is consistent with primarily activation of group Ia fibers, although a contribution from group Ib afferents (Golgi tendon organs) cannot be entirely ruled out. At the cervical level, transmission within the propriospinal system is known to exhibit hemispheric asymmetry associated with hand dominance (Marchand-Pauvert et al., 1999a); the only spinal circuit that, to our knowledge, exhibits lateralization related to hand dominance (Pierrot-Deseilligny and Burke, 2005). In the present study, altered activation of this system was specifically observed on the dominant side in patients; a finding that likely reflects both the lateralized nature of cervical propriospinal connectivity and the well-documented tendency for ALS symptoms to begin asymmetrically (Benetton et al., 2025; Turner et al., 2011). However, the relationship between this lateralization, limb dominance, and side of clinical onset could not be firmly established, likely due to clinical heterogeneity and experimental constraints within the present cohort. Excitatory transmission through cervical and lumbar propriospinal neurons is under strong inhibitory control, primarily mediated by interneurons that receive converging descending (including corticospinal) and sensory inputs, particularly cutaneous afferents (Marchand-Pauvert et al., 2002; Nicolas et al., 2001; Nielsen and Pierrot-Deseilligny, 1991). This inhibitory influence is so robust that it can suppress propriospinal excitation and prevent its transmission to

motoneurons. While the increased propriospinal excitation observed in patients could have been attributed to a reduction in this inhibitory control, the lack of modulation in the corresponding inhibitory pathways suggests that the enhancement of propriospinal excitation is more likely due to a primary alteration of the excitatory propriospinal pathway itself.

The D1 inhibition method is a classical approach for assessing presynaptic inhibition of group Ia afferents projecting directly onto spinal motoneurons (Pierrot-Deseilligny and Burke, 2005). At the lumbar level, presynaptic inhibition is known to be markedly reduced in ALS patients (Castro et al., 2024; Howells et al., 2020; Morin and Pierrot-Deseilligny, 1988; Schieppati et al., 1985). The present study demonstrates that its cervical counterpart is also decreased, suggesting that reduced presynaptic inhibition of the group Ia-motoneuron synapse is likely a generalized feature of ALS pathophysiology and may contribute to hyperreflexia (Sangari et al., 2024). The precise mechanisms underlying presynaptic inhibition at the lumbar level has been challenged by recent findings suggesting that primary afferent depolarization (PAD), mediated by GABAergic interneurons acting on axonal GABA-A receptors, plays a major role in modulating group Ia afferents (Metz et al., 2023). However, the PAD-induced suppression emerged at ISIs (> 60–80 ms), whereas the present protocol used shorter ISIs (21 ms), suggesting that distinct (interneurons acting at presynaptic level and GABAergic axonal receptors) but possibly converging inhibitory processes may be involved.

The postsynaptic inhibition observed with the D1 protocol at shorter ISI than presynaptic inhibition is typically considered to reflect reciprocal inhibition between antagonistic muscles, mediated by Ia inhibitory interneurons. Preliminary results had previously suggested that this pathway might be impaired at the lumbar level (Howells et al., 2020), a finding confirmed by the present study (Supplementary Fig. S2). In contrast, we found no evidence of altered reciprocal inhibition at the cervical level. This absence of change is likely due to the fact that inhibition between wrist flexors and extensors is not mediated solely by reciprocal inhibition, but rather involves a broader class of interneurons responsible for non-reciprocal group I inhibition. These interneurons are
commonly referred to as Ib interneurons (predominant group Ib inputs). However, they also receive converging input from group Ia, group II, joint, and cutaneous afferents (Wagon et al., 2006). This result aligns with our findings at lumbar level, showing that propriospinal excitations are similarly suppressed by the activation of Ib inhibitory interneurons during voluntary contraction in both ALS patients and controls (Marchand-Pauvert et al., 2002). It is also consistent with the absence of any changes in classical group Ib inhibition, as assessed via the modulation of the soleus H-reflex by stimulation of the GM and sural nerves. Altogether, these converging results suggest that, unlike presynaptic or postsynaptic group Ia inhibition, Ib inhibition is largely preserved in the present group of ALS patients.

Cutaneomuscular reflexes, whether the initial excitatory phase (E1) or the subsequent inhibitory phase (I1), did not differ significantly between patients and controls, either in their temporal characteristics or in the strength of the effects. Moreover, the paradigm used in this study has previously been applied to patients with spasticity following spinal cord injury, where it served as a tool to assess motoneuron excitability. In that context, the emergence and persistence of LLR have been interpreted as evidence for motoneuron hyperexcitability, potentially mediated by enhanced persistent inward currents (D'Amico et al., 2013). The absence of such LLR in ALS patients thus provides further support for the view that spinal motoneurons are not intrinsically hyperexcitable in ALS, in line with previous findings (Delestrée et al., 2014; Devlin et al., 2015; Marchand-Pauvert et al., 2019).

Taken together, these findings suggest that spinal circuits primarily driven by muscle spindle afferents are particularly susceptible to alteration in ALS, indicating a selective rather than global reorganization of spinal microcircuits. In addition, these results raise the question of whether the observed imbalance primarily reflects enhanced spinal excitation as reported at the cortical level, with intracortical facilitation often more markedly increased than inhibition is decreased, particularly in specific ALS phenotypes (Tankisi et al., 2025; Van den Bos et al., 2018). However, previous studies have reported alterations in cutaneomuscular (inhibitory) reflexes in ALS patients (Castro et al., 2024; Castro et al., 2021). A possible explanation for the apparent discrepancy with the present findings may lie in the extensive peripheral convergence onto the spinal interneurons, including those involved in transmitting cutaneomuscular responses via flexor reflex afferent (FRA) interneurons. FRA interneurons receive converging input from nearly all classes of sensory afferents, except group Ia (Jankowska, 1992; Pierrot-Deseilligny and Burke, 2005). Given the substantial anatomical variability in peripheral nerve organization across individuals, the resulting sensitivity of spinal responses to electrical stimulation, and the clinical heterogeneity of ALS in terms of phenotype and disease stage, divergent results across studies are to be expected.

To account for clinical variability, we compared each patient's individual response to the $CI_{95}$ of the control group, allowing us to reassess circuit behaviour in relation to clinical status. The results suggest that spinal circuit impairment in ALS follows a temporally structured trajectory, with inhibitory dysfunction emerging early in the disease course, while excitatory enhancement becomes more prominent as the disease progresses. Moreover, this analysis further confirmed that the spinal alterations are afferent-driven, with interneuron microcircuits primarily activated by muscle spindle afferents showing the highest susceptibility to pathological modulation. Importantly, this preferential vulnerability does not exclude the involvement of other spinal circuits, driven by afferents from other sources, as the extensive convergence of sensory inputs and the progressive nature of ALS may ultimately lead to broader network recruitment over time.

## 4.2. Interneuron dysfunction: cellular or network-driven

The spinal alterations in ALS likely result from distinct but interacting mechanisms, including altered corticospinal and/or peripheral drive, intrinsic interneuron dysfunction, and maladaptive plasticity

within sensorimotor circuits. Abnormal descending input from the motor cortex, reflected here by increased AMT and reduced MEPs (Sangari et al., 2024), may contribute to spinal dysfunctions. However, given the diffuse nature of corticospinal projections onto spinal circuitries, descending impairment alone cannot account for the specific and selective alterations of spinal circuitries. As for peripheral drive, sensory axon excitability appears to remain largely preserved, particularly during early stages of ALS (Matamala et al., 2018; Stephanova et al., 2001), making it unlikely that the afferent volleys generated by electrical stimulation differed substantially between patients and controls. If anything, the increased perceptual sensitivity occasionally observed in patients was not associated with measurable changes in spinal responses.

The observation that spinal circuits primarily activated by muscle spindle afferents are the most disrupted in ALS is consistent with findings from animal models. Muscle spindles receive motor innervation from γ-motoneurons, which one might expect to be affected in motor neuron disease. However, γ-motoneurons are largely spared in preclinical models of ALS. This resistance may stem from their lack of direct monosynaptic input from group Ia afferents, a feature they share with other resilient motoneuron populations including oculomotor neurons (Lalancette-Hebert et al., 2016). Paradoxically, γ-motoneurons may indirectly contribute to α-motoneuron degeneration by sustaining excessive group Ia afferent discharge, particularly in the context of disease-related reductions in Renshaw cell activation (Castro et al., 2024; Özyurt et al., 2020; Sangari et al., 2022), releasing γ-motoneurons from recurrent inhibition (Jankowska and Smith, 1973). Accordingly, genetic ablation of group Ia afferents or γ-motoneurons in ALS mouse models delays disease onset and prolongs survival, supporting a pathogenic role for persistent spindle-driven excitation (Lalancette-Hebert et al., 2016). Nonetheless, several lines of evidence indicate that this mechanism alone cannot fully account for the alterations observed in the present study. First, muscle spindles undergo early structural disorganization in SOD1 mice, and their sensory endings degenerate alongside motor axons, although their decline is slower (Vaughan et al., 2015). This suggests that any early group Ia excitatory overdrive may be transient, eventually giving way to afferent input failure as the disease progresses. Second, the group Ia-motoneuron synapse itself appears functionally weak and structurally unstable in ALS mice (Bączyk et al., 2020), raising the possibility that intrinsic synaptic fragility contributes to early circuit vulnerability. However, reduced presynaptic inhibitory controls reported in humans (Castro et al., 2024; Howells et al., 2020; Morin and Pierrot-Deseilligny, 1988; Sangari et al., 2016; Schieppati et al., 1985), including post activation depression (Özyurt et al., 2020), may counteract the expected decline in synaptic efficacy, thereby maintaining excessive excitation (Sangari et al., 2024). Third, the closely linked distribution of group Ia afferent projections and Renshaw cell-mediated inhibition varies regionally, which may contribute to the differential ALS-related sensitivity of hindlimb muscles in mice (Lalancette-Hebert et al., 2016). However, this explanation is less applicable to humans, where these combined connections are more prominent at the lumbar level and markedly reduced in the cervical cord, compared to animal models (adaptation to bipedal locomotion) (Pierrot-Deseilligny and Burke, 2005). Although differences in ALS vulnerability exist between individual muscles in humans (distal vs. proximal muscles), upper and lower limb musculature is, overall, affected to a comparable degree.

Altogether, while the afferent-driven model provides a compelling framework for interpreting circuit vulnerability, it is unlikely to represent the sole mechanism underlying spinal dysfunction in ALS. In support of this, preclinical studies have begun to clarify the role of spinal interneurons in ALS pathophysiology. A consistent finding across studies is that inhibitory interneurons are particularly vulnerable, showing signs of dysfunction even before LMN degeneration. This includes glycinergic and GABAergic populations, such as presumed Ia inhibitory interneurons and Renshaw cells (V1 interneurons), whose early loss may
underlie the widespread reduction in spinal inhibition reported in both animal models and humans (Castro et al., 2024; Castro et al., 2021; Howells et al., 2020; Montañana-Rosell et al., 2024; Özyurt et al., 2020; Sangari et al., 2022). In contrast, excitatory interneurons, including glutamatergic V2a interneurons, appear more resilient at disease onset but undergo later functional or structural changes that may contribute to dysregulated spinal output as ALS progresses (Goffin et al., 2024; Montañana-Rosell et al., 2024). These changes are accompanied by multiphasic homeostatic adaptations at the circuit level, including early synaptic scaling and intrinsic plasticity, followed by a collapse of compensatory balance as LMN degeneration advances (Nascimento et al., 2024). This dynamic pattern mirrors the early inhibitory deficits and later excitatory amplification observed in the present study.

Beyond the excitatory/inhibitory framework, growing attention is being paid to the role of neuromodulatory systems (Scekic-Zahirovic et al., 2024). In line with this, cholinergic interneurons (V0c), which regulate motor output gain via presynaptic mechanisms, are implicated in ALS-related spinal circuit disturbances (Goffin et al., 2024). Although their involvement remains to be demonstrated in humans, altered neuromodulatory tone could further destabilize spinal dynamics by amplifying existing excitation/inhibition imbalances. This possibility is supported by the present findings, as lumbar interneurons receiving group I and group II inputs are known to be highly sensitive to monoaminergic modulation (Lourenço et al., 2006; Maupas et al., 2004; Rémy-Néris et al., 2003).

Several conceptual limitations must be considered when interpreting these findings. First, the precise identity of spinal interneuron subtypes in rodents remains largely inferred from developmental lineage markers and shaped by phylogenetic evolution, complicating direct comparisons with functionally defined interneurons and pathophysiological processes in humans (Sengupta and Bagnall, 2023). However, previous studies (Castro et al., 2024; Özyurt et al., 2020; Sangari et al., 2022), along with the present results, suggest some correspondence, particularly for Ia inhibitory interneurons and Renshaw cells, which consistently show early vulnerability. Second, most ALS models emphasize cell-autonomous mechanisms, often overlooking the critical contribution of structured sensory and descending inputs to the development and maintenance of spinal interneurons (Plant et al., 2018; Smith et al., 2017). Since these interneurons rely on patterned afferent activity for proper maturation, early disruptions, such as spindle disorganization (Vaughan et al., 2015) or improperly configured corticospinal projections during development (Özdinler et al., 2011; Scekic-Zahirovic et al., 2021), may initiate or accelerate their dysfunction. Finally, while early loss of inhibitory interneurons is now well documented, far more so than changes in excitatory interneurons, the downstream consequences for motor control, compensatory reorganization, and the spread of dysfunction across spinal networks remain poorly understood.

Taken together, these findings suggest that ALS-related spinal circuit dysfunction reflects a complex interplay between afferent input disruption, interneuron vulnerability, and maladaptive plasticity. The present human data support this framework, revealing a temporally organized sequence in which inhibitory dysfunction precedes excitatory amplification, particularly in circuits driven by muscle spindle afferents, consistent with a convergence of interneuronopathy and afferent-driven mechanisms in ALS.

### 4.3. Involvement in ALS pathophysiology and therapeutic perspectives

Hyperexcitability has been consistently observed in both animal models and human studies, making it a hallmark feature of ALS. Notably, a reduction in intracortical inhibition has been identified as one of the earliest pathophysiological changes, even at presymptomatic stages of the disease, suggesting early impairment of GABAergic inhibitory interneurons in the motor cortex (Gunes et al., 2020; Higashihara et al., 2024). As ALS progresses, particularly in specific phenotypes, this early inhibitory deficit is often followed by a progressive increase in

intracortical facilitation, reflecting a growing imbalance in cortical excitatory/inhibitory dynamics (Pavey et al., 2024; Van den Bos et al., 2018). More recent work conceptualizes these cortical alterations within a dynamic excitability continuum, in which early hyperexcitability may evolve toward hypoexcitability or inexcitability as corticospinal degeneration advances, with important prognostic implications (Ranieri et al., 2025; Ranieri et al., 2021). The present study, along with recent findings from preclinical models (Goffin et al., 2024; Montañana-Rosell et al., 2024), suggests that a comparable temporally structured sequence may also occur at the spinal level, potentially mirroring and contributing to the broader alterations observed across motor networks in ALS. With disease progression, motoneuron loss and degeneration of spinal output pathways are expected to reduce the overall gain of spinal motor circuits, leading to an apparent normalization and eventually to hypoexcitability, as suggested by reduced H-reflex amplitudes and preserved or reduced intrinsic motoneuron excitability reported in ALS patients (Marchand-Pauvert et al., 2019; Sangari et al., 2024).

This raises the question of whether cortical and spinal circuit alterations evolve independently or share common mechanisms. While sensory integration is also altered cortically in ALS, no specific afferent-driven pattern comparable to the one observed here in the spinal cord has been reported (Kleinerova et al., 2025). A likely reason is that sensory signals undergo extensive filtering and modulation at subcortical levels before reaching the cerebral cortex, thereby limiting a direct influence of peripheral afferent input on cortical sensory responses. Moreover, cortical interneurons are predominantly inhibitory and function mainly to fine-tune local network activity (Gunes et al., 2020; Rosés-Novella and Bernard, 2025), whereas spinal networks comprise both inhibitory and excitatory interneurons organized into tightly coupled microcircuits with distinct roles in shaping sensorimotor integration and reflex control (Alaynick et al., 2011; Jankowska, 1992; Pierrot-Deseilligny and Burke, 2005). The only evident network-driven link between cortical and spinal dysfunction lies in the early cortical impairments supporting the corticofugal hypothesis (Higashihara et al., 2024; Scekic-Zahirovic et al., 2021), whereby cortical hyperexcitability may propagate along descending pathways, potentially involving extrapyramidal tracts as well (Christidi et al., 2018), and influence spinal network stability. However, this mechanism alone cannot fully account for the spinal circuit disruption, particularly the dynamic interplay between inhibitory and excitatory defects and the afferent-driven nature of the alterations. Together, these findings suggest that while corticospinal dysfunction likely contributes to spinal alterations, distinct and partially independent degenerative processes are also at play. In particular, muscle spindle-driven spinal dysfunctions may underlie specific clinical phenotypes and likely contribute to the heterogeneity of ALS presentations, including variability in disease onset, propagation, and predominant motor neuron involvement (UMN, LMN, or mixed forms).

Spinal interneurons operate within highly interactive circuits, where inhibitory and excitatory elements dynamically balance motoneuron output. This structural organization raises the possibility that early loss or dysfunction of inhibitory interneurons, together with reduced corticospinal input, may progressively destabilize excitatory circuits. Disinhibition could drive maladaptive plasticity or hyperactivity in excitatory interneurons, ultimately amplifying motoneuron activation and contributing to the later-emerging excitatory overdrive observed in ALS. For instance, the consistent reduction in presynaptic inhibition of group Ia terminals observed in ALS could facilitate excessive excitation of propriospinal pathways (Burke et al., 1992a). In this context, the absence of propriospinal inputs to intrinsic hand muscles may contribute to the high vulnerability of these motoneuron pools (Supplementary Fig. S5), while the ability of propriospinal pathways to bypass the orderly recruitment described by the Henneman size principle could favour early activation of fast-fatigable motoneurons (Marchand-Pauvert et al., 2000), which are known to be particularly susceptible in ALS (Fogarty et al., 2020). These considerations should be viewed as mechanistic hypotheses requiring further experimental validation.
From a translational perspective, the present findings do not identify direct therapeutic targets but highlight spinal circuit components whose dysfunction may contribute to motor network imbalance in ALS. In this context, neuromodulation approaches such as non-invasive brain stimulation and transcutaneous spinal direct current stimulation represent promising strategies to influence motor system excitability by acting on distributed cortical and spinal networks, including interneuronal circuits. Emerging evidence from human studies, together with supportive data from animal models, suggests that these approaches can induce measurable changes in motor network excitability and plasticity (Bączyk et al., 2021; Di Lazzaro et al., 2024; Jankowiak et al., 2022). While their clinical efficacy in ALS remains to be fully established, current data support the view that neuromodulation may have therapeutic potential, particularly if interventions are tailored to disease stage and clinical phenotype. By contrast, riluzole treatment did not show a measurable association with spinal excitability measures in the present cohort, consistent with previous reports indicating that its modulatory effects on neural excitability in humans are modest and transient (Geevasinga et al., 2016). Future work combining detailed neurophysiological characterization with pharmacological and neuromodulation protocols will be essential to determine how these approaches can be optimally personalized and integrated into therapeutic strategies for ALS.

## 5. Limitations and future directions

Together, our findings reveal a structured disruption of spinal microcircuits in ALS. However, several limitations must be acknowledged. First, not all spinal circuits were assessed in every participant, although the high degree of overlap between tested pathways supports the consistency of the findings. Second, the cross-sectional design does not allow for intra-individual temporal tracking of circuit dynamics, underscoring the need for longitudinal studies, though such follow-up is practically challenging in this population. In addition, although disease stage and functional impairment were incorporated into the analyses, we did not stratify patients according to detailed clinical phenotypes. Given the strong phenotype-dependent modulation of excitability reported at the cortical level (Ranieri et al., 2025), future studies will need to determine whether similar phenotype-specific trajectories also exist at the spinal level, ideally using larger and longitudinal cohorts. Third, while our results support an afferent-specific pattern of dysfunction, the possibility of convergence between circuits (e.g., via shared interneurons) limits definitive conclusions about strict input selectivity. Finally, preclinical models and computational simulations will be essential to test the 'afferent-driven circuit vulnerability' hypothesis in greater mechanistic detail and to identify cell-type-specific targets for future therapeutic interventions.

While such mechanistic insights cannot yet be directly tested in humans, the convergence between our findings and preclinical models underscores the need for further research into dynamic alterations within spinal circuits. A deeper understanding of these changes could shed light on fundamental aspects of ALS pathophysiology and open new therapeutic avenues targeting specific neuronal subtypes. Additionally, the selective vulnerability of circuits driven by muscle spindle afferents highlights the potential for sensorimotor rehabilitation strategies or targeted neuromodulation approaches to promote neuroprotection and slow disease progression (Banos et al., 2025).

### CRediT authorship contribution statement

**Sina Sangari:** Writing – review & editing, Investigation, Formal analysis.
**Alexandra Lackmy-Vallée:** Writing – review & editing, Validation, Supervision, Methodology, Investigation, Formal analysis.
**Ise-line Peyre:** Writing – review & editing, Investigation, Formal analysis.
**Pierre-François Pradat:** Writing – review & editing, Validation, Supervision, Methodology, Investigation, Formal analysis, Conceptualization.
**Véronique Marchand-Pauvert:** Writing – review & editing,

Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

### Declaration of competing interest

The authors declare that they have no competing interests.

### Acknowledgments

The authors thank all the participants and the Paris ALS Referent Center at Pitié-Salpetrière Hospital for their support in patient selection. They are also grateful to Genethon (Evry, France) and the University of Tours (Prof. P. Vourc'h, France) for performing DNA extraction and analysis. This work was generously supported by grants from ARSLA (VMarchand/2013), AFM-Téléthon (DdT1 2015-2; CTL/SS/2016-0029/ no. 16597), and the Fondation Thierry Latran (FTL AAP7/2015). During his PhD, supervised by V. Marchand-Pauvert, S. Sangari received financial support from the French Ministry of Higher Education, Research and Innovation via a doctoral grant from Université Pierre et Marie Curie – Paris 6 (now Sorbonne University; 2012–2015), and from AFM-Téléthon (2015–2016). During his PhD, he also received funding from the “Plan d'Investissement d'Avenir” (PIA) via the IdEx Sorbonne Universités program (SUPER), which supported his research stay supervised by Pr. D. Burke, at the Brain and Mind Centre (University of Sydney).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.nbd.2026.107270.

### Data availability

Data will be made available on request.

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