# Reactive and adaptive remodeling of S1 cortical maps correlates with functional deficits and recovery following M1 microinfarct

Fatemehsadat Abtahi, Yoh'i Zennou-Azogui, Sabine Quitard, Christian Xerri

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Fatemehsadat Abtahi, Yoh'i Zennou-Azogui, Sabine Quitard, Christian Xerri. Reactive and adaptive remodeling of S1 cortical maps correlates with functional deficits and recovery following M1 microinfarct. Neuroscience, 2025, 582, pp.146 - 163. 10.1016/j.neuroscience.2025.07.034. hal-05290648

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Research Article

Reactive and adaptive remodeling of S1 cortical maps correlates with
functional deficits and recovery following M1 microinfarct

Fatemehsadat Abtahi, Yoh'i Zennou-Azogui, Sabine Quitard, Christian Xerri

Aix-Marseille University - CNRS, Center for Research in Psychology and Neuroscience (CRPN), UMR 7077, Marseille, France

ARTICLE INFO

Keywords:

Receptive field dynamics
Somatosensory cortical mapping
Cortical reorganization
Sensorimotor integration
Fine sensorimotor skills
Rehabilitation-enhanced plasticity

A B S T R A C T

Reciprocal connections between the S1 and M1 cortices suggest a crucial role for somatosensory processing in cortical reorganization following M1 injury. However, the specific contribution of S1 to functional recovery after M1 lesions remains unclear. Here, we investigated remodeling of the S1 forepaw map alongside spontaneous sensorimotor recovery following a focal lesion to the caudal forelimb area (CFA) of M1. We also evaluated how a rehabilitation protocol influenced both cortical reorganization and behavioral outcomes. A microinfarct in the CFA induced marked alterations in the ipsilesional S1 forepaw cutaneous map and caused pronounced asymmetries in gross and fine forelimb sensorimotor tasks. These deficits were transient, with full recovery occurring within two months and significantly accelerated by rehabilitation. The cortical representation of the ventral skin gradually expanded beyond its pre-lesion size in association with behavioral recovery, while the representation of the dorsal skin remained unchanged. Initially, the lesion caused an expansion of both ventral and dorsal skin receptive fields (RFs), peaking at 14 days postlesion. Ventral RFs subsequently refined, becoming smaller than pre-lesion size, whereas dorsal RFs returned to baseline. Early rehabilitation accelerated RF shrinkage, particularly in ventral areas, mirroring late-stage spontaneous recovery. Neuronal sensitivity to light touch increased steadily post-lesion, with rehabilitation shortening this progression. The cutaneous RFs' sharpening and ventral cortical territory expansion closely paralleled recovery of fine sensorimotor skills. These findings highlight the pivotal role of S1 cortical plasticity in sensorimotor recovery following M1 injury and support early rehabilitation as a facilitator of adaptive cortical reorganization and functional recovery.

Introduction

Sensorimotor integration is underpinned by an ongoing crosstalk
between somatosensory and motor cortical areas. This process is crucial
for generating skilled movements and adaptive motor control through
feedforward motor commands and sensory feedback (Johansson and
Cole, 1992; Lam et al., 2006). Therefore, improvement of behavioural
deficits induced by motor cortex lesion requires an adjustment of both
predictive and corrective sensorimotor control for gradually recalci-
brating motor output (Borich et al., 2015; Bolognini et al., 2016).

Reciprocal connections between primary somatosensory (S1) and
motor (M1) cortices (Rocco-Donovan et al., 2011; Petrof et al., 2015),
situated at the core of a distributed cortical and subcortical network,
suggest that somatosensory processing may crucially contribute to
cortical reorganization following M1 injury. Numerous studies have
thoroughly investigated the anatomical and functional cortical

alterations, aiming to uncover their potential role in functional resto-
ration (for review, see Nudo, 2003; Ward, 2011; Jones and Adkins,
2015). The role of the ipsilesional motor cortex in recovery following
stroke in M1 has been extensively investigated in both patients (Weiller
et al., 1992; Cicinelli et al., 1997; Traversa et al., 1997; Hallett, 2001)
and animal stroke models (Glees et al., 1950; Castro-Alamancos and
Borrell, 1995; Liu and Rouiller, 1999; Jones and Adkins, 2015). By
contrast, despite a consensus about the importance of sensory integra-
tion in motor control and the well-documented lesion-induced cortical
plasticity, the contribution of the S1 cortex to the recovery of skilled
movement after motor cortex injury remains relatively unexplored (Paul
et al., 2006; Harrison et al., 2013; Fukui et al., 2020). In particular, no
longitudinal study had yet provided evidence for post-lesion changes in
neuronal receptive fields or responsiveness in S1 following focal M1
injury, features critical for tactile discrimination and manual dexterity,
or for how rehabilitation training might influence such changes.

<footnote>* Corresponding author at: Aix-Marseille Université-Site Saint Charles, 3 place Victor Hugo, 13331 Marseille, Cedex 3, France.</footnote>

<footnote>E-mail addresses: fatemehsadat.abtahi@univ-amu.fr (F. Abtahi), yohi.zennou-azogui@univ-amu.fr (Y. Zennou-Azogui), sabine.quitard@univ-amu.fr (S. Quitard), christian.xerri@univ-amu.fr (C. Xerri).</footnote>
Therefore, the primary objective of the present investigation was to gain insight into the temporal dynamics of changes in the forepaw cutaneous area in S1, along with sensorimotor deficits following a lesion confined to the forepaw representational region within the caudal forelimb area (CFA) of M1. We reasoned that following M1 injury, immediate changes in S1 would reveal important characteristics of M1-S1 interactions in the context of *reactive neuroplasticity* which, in our view, refers to the spontaneous, injury-induced reorganization, often involving the rapid unmasking of latent connections or the expansion of adjacent areas into damaged regions. These early changes reflect disrupted function and underlying neural organization, but are not always beneficial. We hypothesized that increasing reliance on somatosensory feedback may compensate for deficits in fine sensorimotor control, thereby promoting adaptive changes in the response properties of S1 neurons. In our view, *adaptive neuroplasticity* involves experience- and training-dependent refinement of cortical maps, shaped by task demands and sensorimotor activity. This form of reorganization supports the recovery of skilled behaviour by promoting efficient remodelling of functional representations.

Consistently, a wealth of studies on cortical plasticity have established that somatosensory cortical maps are experience-dependent and dynamically maintained representations that play a major role in processing of behaviourally relevant sensory inputs underlying the acquisition of sensorimotor skills (for review, see *Xerri, 2008*). Historically, this discovery has given powerful impetus to the therapeutic implications of experience-dependent cortical plasticity following brain injury. Previous studies have separately investigated the impact of rehabilitative training on postlesion changes taking place in M1 or S1. Interestingly, learning skilled hand use prevents the loss of the trained-hand representation in the territory adjacent to the M1 infarct, and promotes the recovery of manual dexterity (Nudo and Milliken, 1996). Similarly, an enriched environment and tactile stimulation drive a neuroprotective process that preserves the somatosensory representations in perilesion zones following S1 focal infarct (Xerri et al., 2003; Xerri and Zennou-Azogui, 2014).

The present study lies at the intersection of these two distinct, yet converging, lines of research on postlesion plasticity of somatosensory or motor cortical representations. We addressed how sensorimotor rehabilitation might drive adaptive changes in S1 following M1 focal injury. Therefore, the second aim of the present study was to analyse the effects of an early rehabilitative procedure on the time-course of the recovery of sensorimotor abilities and correlative cortical map remodelling in the S1 forepaw representation after M1 lesion. We hypothesized that reinforcing somatosensory feedback and sensorimotor experience during an early period known to be critical for postlesion plasticity would generate cortical changes likely to improve and accelerate functional recovery.

## Experimental procedures

All experiments were performed in accordance with the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. The present study was approved by Neurosciences Ethics Committee N° 71 of the French National Committee of Animal Experimentation. Each step of these experiments, from conception to implementation, was carried out under the supervision of a veterinarian and in agreement with the National Ethics Committee. Every attempt was made to respect the 3Rs principle to minimize both the number, the distress and the suffering of the animals used in this experiment.

## Animal housing and care

Animals were housed in groups of three in Plexiglas cages (26.5 cm wide, 42.5 cm deep, and 18 cm high). The floors of all cages were covered with sawdust. All animals were maintained on a 12-hour light/

dark cycle, at a constant temperature (24 ± 1 °C) with approximately 60 % relative humidity. Food and water were provided *ad libitum*. However, to determine the rats' hand preference, we used a food pellet grasping task during a three-week period while the animals were subjected to a food restriction, receiving 85 % of their daily food intake.

## Experimental groups

In this study, three-month-old male Long-Evans rats (n = 52), weighing between 350 and 400 g, were obtained from Charles River Laboratory (Evreux, France). The animals were randomly assigned to 7 experimental groups: *Control rats* (n = 8) were used for comparing cortical mapping data with that of lesioned rats; *Acute rats* (n = 8) underwent a focal infarct in M1 and electrophysiological mapping in S1 were performed one hour after the lesion induction; *2-week rats* (n = 8) and *5-week rats* (n = 8) had cortical mapping 14 days and 35 days after the lesion, respectively. In addition, *2-week + R rats* (n = 8) were subjected to cortical mapping after they had received three rehabilitative sessions during each of the two weeks post-lesion. In the animals allowed to recover from the lesion, behavioural testing was repeated three times per week, for two weeks prior to the lesion and during the subsequent weeks until cortical mapping was performed. We also included a group of rats that did not experience regular behavioural assessment over the postoperative time but were subjected to two evaluations prior to the lesion and within the 8th week postlesion, before cortical mapping (*8-week rats*, n = 8). In *sham rats* (n = 4), the surgical procedure was performed, except for the cortical lesion, and behavioural testing was carried out on days 3 and 6 postlesion. The experimental groups were labelled as follows: *Control, Acute, 2-W, 2-W + R, 5-W, 8-W* and *Sham* (see Fig. 1A for experimental design overview).

## Surgical procedures

Animals were anesthetized by intraperitoneal injection of ketamine 1000 (25 mg/kg, Virbac, France) and medetomidine (0.25 mg/kg, Domitor® OrionPharma, Finland). Anesthesia was continued throughout the procedure by administering half the initial dose of this mixture every hour to obtain a stable level of anesthesia. A local anesthetic (Xylocaine, Xylovet®) was applied to the scalp and external auditory meatus to minimize the pain generated by the skin incision and the ear bars. The head was held in a stereotaxic frame at the level of the incisors and the ear bars. Body temperature was continuously monitored by a rectal thermo probe and maintained between 37.5 °C and 38 °C by a homeothermic feedback blanket (Harvard Apparatus Ltd., Kent, UK). The animal was given a subcutaneous injection (2.5 ml) of a mixture of equal parts NaCl and glucose (5 %) which was renewed every 2 h to maintain the animal in a stable physiological state.

The surgical and lesioning procedures were performed under aseptic conditions. To prevent cerebral edema, an incision was made between the first vertebra and the skull, and after resection of posterior neck muscles, the cerebrospinal fluid was drained through an opening in the dura mater overlying the *foramen magnum*. The scalp was then incised, and after detaching the connective tissue and lateral muscles overlying the parietal bone, a craniotomy was made between +3 and -4 mm from bregma in the rostro-caudal axis and between 1 and 6 mm in the mediolateral direction to expose both the S1 and M1 forelimb areas. The removed bone flap was kept hydrated in NaCl so that it could be placed back at the end of the procedure. After incising and resecting the dura mater, the surface of the exposed parietal cortex was bathed in a thin layer of warm silicone fluid (30,000 cenistokes; Accumetrics) to prevent drying and oedema. In all lesioned rats except the *Acute* group, a local antibiotic (Flumiquil®, Ceva Santé Animale) was applied after the lesion induction on the exposed cortex which was then covered with gelatin film (Gelfilm, Upjohn). The bone flap was replaced, then stabilized and sealed with dental cement before the neck muscles and scalp were sewn back together. A non-steroidal anti-inflammatory analgesic was
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Experimental protocol for evaluating sensorimotor deficits and remodeling of the forepaw cutaneous map in S1 after a focal lesion in the caudal forelimb area (CFA) in M1. (A) Training was conducted to assess forelimb preference during a pellet retrieval task. Baseline performance was assessed at 3 and 1 days before the cortical lesion in the 2-W, 2-W + R, 5-W, and 8-W groups, and was resumed three times per week after the lesion and continued until the cortical mapping session. The timeline of experiments is indicated for each group, with color-coded lines referring to behavioural assessment periods. The cortical lesion was induced on Day 0 (vertical red bar). Downward arrows indicate the time points at which electrophysiological mapping was performed. Triangles mark the time of brain perfusion/extraction for histological analysis of lesion volume. (B) Schematic representation of the cortical lesion procedure. A thermal probe was positioned stereotactically over the caudal forelimb area (CFA) in M1 to produce a focal ischemic injury in the functionally identified forepaw representational zone (broken line circles). (C) Electrophysiological mapping: 1. Recordings were performed in layer IV of the S1 forepaw area (FA). Approximately 100 cortical sites were recorded per mapping to assess the location and size of neurons’ cutaneous receptive fields (RFs), as well as their responsiveness to skin stimulation (black rectangles refer to unresponsive sites; black dots indicate non cutaneous responses); 2. RFs were classified based on their location on the forepaw (digits 1–5 and palm); 3. Representative examples of ventral RFs recorded during a single mapping session; 4. These RFs were used to generate a somatotopic map of the S1 forepaw representation, distinguishing glabrous ventral (light colors) from hairy dorsal (dark colors) skin surfaces.</figcaption></figure>
administered before the animal regained consciousness (Tolfedine, Vétiquinol, 4 mg/kg, i.m.). Antibiotic prophylaxis (ceftazidime; 15 mg/kg) was used before the beginning, at the end and the day after the surgery, in order to avoid the development of a brain infection.

The injured rat was then placed in a post-operative room under increased surveillance for the duration of recovery from surgery. The animal was kept alone in a cage with jelly food and water ad libitum for 2 days, then was returned to standard housing conditions with two cage mates. The 2-W, 2-W + R, 5-W and 8-W rats were re-anesthetized after completion of their respective sessions of behavioural assessment and S1 mapping was performed after removal of the sealed bone flap and protective gelatin film.

## Induction of cortical lesion

The lesion targeted the CFA in the hemisphere contralateral to the dominant forepaw, previously assessed with a food reaching task. The silicone fluid was removed, and the exposed cortex was bathed in warm physiological saline. A temperature-controlled electrode (Radionics TCZ; Kopf; USA) was moved perpendicular to the cortex surface by the micromanipulator and was slowly lowered so that its tip (diameter: 0.25 mm) was in contact with the cortical surface within the functionally identified forepaw region of the CFA. A 500 MHz radiofrequency current was delivered through the electrode, and the temperature measured at its tip, equipped with a thermistor, was gradually raised to 80 °C within 1 min and maintained at this level for 3 min. The lesion was induced at two very close locations of the CFA while carefully avoiding proximity with the somatosensory forepaw area (Fig. 1B). In addition to direct destruction of the neural tissue, the heat generated in the brain tissue induced a focal infarct, characterized by a visible occlusion of the vessels, along with a blanching of the cortical zone around the electrode tip. Care was taken to preserve major arteries and veins while occluding their local branches. Radiofrequency-induced hyperthermia produces cellular injury, i.e., coagulation necrosis, neuronal shrinkage, nuclear pyknosis and perineuronal astrocytic swelling (Ohmoto et al., 1996), which is associated with cerebral ischemia. Moreover, localized hyperthermia induces increased extracellular glutamate concentrations that reaches neurotoxic levels (Adachi et al., 1995).

## General experimental procedure for electrophysiological data collection

As a preliminary step, a small sample of prelesion data (cutaneous receptive fields and mechanical threshold of responses) was collected from 15 to 20 cortical sites in S1 by recording somatosensory potentials evoked by the contralateral forepaw tactile stimulation. Afterward, we used intracortical microstimulation (ICMS) to functionally locate the forepaw region of the caudal forelimb area (CFA) in M1 based on approximately 10 stimulation sites from which low threshold finger or wrist movements were evoked. Then, the lesion was centred on the identified sector of the CFA. In the Acute group, the forepaw mapping in S1 was resumed 1 h after the lesion induction, starting with recordings from the prelesion cortical locations before proceeding with a complete mapping of the forepaw area (see detailed procedures below). In the other lesioned groups, the S1 mapping was performed at the end of the 2nd, 5th, or 8th week postlesion.

## Somatosensory mapping

Conventional multi-unit monitoring and receptive field mapping techniques were used to reconstruct the forepaw representation in layer IV of S1. The mapping procedure used in the present study has been previously described (Xerri, 1999, 2005). We used a high-resolution camera mounted on an operating microscope to obtain magnified images of the cortical area to be mapped and of the ventral and dorsal skin surfaces of the contralateral forepaw. Locations of microelectrode penetrations relative to the vasculature of the cortical surface and

boundaries of cutaneous receptive fields (RFs) were recorded on the digitized images of the cortex and forepaw, respectively, using a home-made program (MATLAB®, Mathworks, Inc.). Small clusters of neurons (usually two to four) were recorded with parylene-coated tungsten microelectrodes (about 1 MΩ at 1 kHz; WPI, UK). The electrode was moved perpendicular to the cortical surface in Cartesian coordinates using a 3-dimensional micromanipulator driven by stepping motors (Märzhauser, FST; Germany). The multi-unit signal was pre-amplified, filtered (bandwidth: 0.5–5 kHz), and displayed on an oscilloscope. This signal was also rectified and passed through a discriminator whose output was proportional to the portion of the input signal that was above an adjustable threshold set just above the background noise. The output of the discriminator was then passed to an audio monitor. Using the artifact generated by the contact of the microelectrode with the surface of the cortex as a zero level, we advanced the electrode to a depth of approximately 650–700 µm to record the responses of small groups of layer IV neurons (Waters et al., 1995; Welker et al., 1984) to forepaw skin stimulation. Under our electrophysiological conditions, the background noise amplitude was generally between 15 and 20 µV with a signal-to-noise ratio between 4 and 6. The interelectrode penetration distance averaged about 150 µm in all groups of rats.

At each recording site, bursts of activity elicited by natural skin stimulation allowed us to classify neuronal responses as cutaneous or non-cutaneous. The cutaneous RFs were defined for each cortical recording site as the area of skin where just-visible skin indentation or hair deflection elicited reliable changes in multiple-unit discharge. This stimulation was produced with a fine-tipped, hand-held glass probe and monitored with 4 × magnifying glasses. The ridges running along the glabrous skin of the digits and palm were used as landmarks to delineate the RFs. These RFs were drawn onto the digitized images of the forepaw ventral and dorsal surfaces recorded for each rat. Responses evoked by nail movements, also classified as cutaneous responses, were examined while the tips of the digits were maintained firmly in place to minimize joint movement and skin deformation. Non-cutaneous responses were identified by taps and pressure on tendons, intrinsic muscle or joint manipulations where no cutaneous response was found. Cortical sites exhibiting only spontaneous discharges were classified as unresponsive.

The mechanical threshold of neural responses to skin stimulation of glabrous skin surfaces was determined using Von Frey monofilaments (Semmes-Weinstein Aesthesiometer, Stoelting, Wood Dale, IL). These filaments apply indentation stimuli at a relatively constant pre-determined bending force. The most commonly used filaments were 2.83 (diameter: 0.127 mm; bending force: 0.07 g; bending pressure: 5.53 g/mm²), 3.22 (diameter: 0.152 mm; bending force: 0.16 g; bending pressure: 8.77 g/mm²), 3.61 (0.178 mm; 0.40 g; 16.10 g/mm²), and 3.84 (0.203 mm; 0.60 g; 18.40 g/mm²). For reproducible measurements, the filaments were used at a relatively constant room temperature (about 24 °C). The stimulation consisted in pressing a filament gently against the skin, perpendicular to its surface at the centre of the RF, until the filament began to bend. This procedure was repeated 5 to 10 times for each filament. We used a stimulus series of increasing and decreasing strengths to determine the mechanical threshold evoking noticeable changes in neuronal discharge. The mechanical thresholds are conventionally used as a measure of the sensitivity of the neuronal response to skin stimulation.

## Measurement of neuronal response characteristics and map features

The size of each cutaneous RF was measured offline using a custom application created with MATLAB® (Mathworks, Inc.). Glabrous (ventral) and hairy (dorsal) skin RFs were expressed in mm² and the average size of all recorded RFs was calculated for each experimental group. We used Canvas X draw software (Canvas GFX, USA) to reconstruct somatotopic maps of the forepaw. Boundaries encompassing cortical sites whose RFs were restricted to a common forepaw subdivision (e.g., finger, palm pad, etc.) were drawn (Fig. 1C). Thus, nearby
cortical sites responding to stimulation of the same skin area were grouped in the same cortical territory of the map. The boundary between two distinct territories was drawn halfway between two adjacent points for which the RFs were restricted to two distinct skin subdivisions of the paw. The same principle was used to draw boundaries encompassing cortical sites where non-cutaneous responses were found for each fore-paw subdivision. The Canvas X Draw software was also used to calculate, in each rat, the whole cutaneous map surface and areal extents (in mm²) of the cortical territories serving the glabrous and hairy skin. Average values were then computed for each experimental group.

Every threshold value of bending pressure measured with the Von

Frey monofilaments was multiplied by the number of recording sites displaying that threshold, and the sum of these weighted values was divided by the total number of sites tested in each animal. A mean threshold was then calculated for each experimental group.

### Motor mapping

Intracortical microstimulation (ICMS) techniques were used to identify and explore the caudal forepaw area (CFA) in M1 contralateral to the preferred forelimb. The digital image of the cortex was used to guide microelectrode penetrations with reference to the surrounding

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Behavioral testing and rehabilitation procedure. (A) Paw preference test: Prior to lesion induction, animals were trained to retrieve food pellets from the center of a platform. The preferred forepaw was identified based on consistent lateralized reaching across 200 trials. (B) Cylinder test: Postural symmetry during spontaneous vertical exploration was assessed by placing the animal in a transparent cylinder. The number of forepaw contacts with the wall during rearing were counted to quantify asymmetry in forelimb use. (C) Ladder walking test: This locomotor task evaluated forepaw placement errors while animals spontaneously traversed upward and downward a 1-meter ladder inclined at 45°. A mirror positioned below the ladder allowed precise visualization of forepaw placement. Video recordings were analysed in slow motion to quantify missteps across five round trips. (D) Schematic representation of a typical training day for the 2-W + R group. The protocol included four 15-minute sessions of imposed walking on a motorized{ forced-locomotion { Carousel } } and 5 minutes of rest. (E) Custom-built motorized { enriched environment } and 30 minutes of texturing the surface of objects composed of various shapes, textures and rugosities. (F) Environment</figcaption></figure>
vasculature. Platinum-iridium microelectrode (100 ΚΩ; WPI, UK), secured to the 3-dimensional manipulator, was lowered to layer V at a depth of about 1600–1700 µm. The stimuli consisted of 60 ms trains of 13 monophasic cathodal pulses (0.2 ms duration; 3.3 ms interpulse interval) with a 1 s interval between trains. This pulse train was triggered through a stimulus pulse generator (Master-8; AMPI, Israel) and delivered by a biphasic stimulus isolator (Model BSI-2; Microprobes for Life Science, USA). Trains were repeated at each cortical site to detect the minimum current level (threshold) required to evoke visible movements. At each electrode penetration, stimulation was initiated at the lowest intensity (5 µA) and was increased gradually until forelimb movement was evoked, up to a maximum of 100 µA. The cortical site was defined as unresponsive when no movement was induced at this maximal value. Limb movements and muscle contractions that were evoked by cortical stimulation were visually detected and confirmed by two observers. Limb posture was adjusted to keep limbs relaxed before stimulation. Our area of interest in the CFA was delineated by defining the borders between sites at which stimulation evoked digit or wrist-forearm movements and sites at which stimulation evoked more proximal (elbow, shoulder, and face) movements or by sites at which stimulation evoked no movement at the maximum current level. The CFA area evoking finger/wrist movements was then targeted to induce a micro-ischemic damage following the procedure described above.

## Behavioral assessment

### Paw preference

Paw preference was determined using a single-pellet reaching test. The testing apparatus consisted of a custom-made plexiglass box (26 cm long × 28.5 cm high × 16 cm wide) with a narrow opening over the entire height in the middle of the front panel. The animal could reach through this opening to retrieve a banana-flavoured pellet (Bioserve, Inc., Frenchtown, NJ) from a food well carved in a plexiglass plate. The opening was small enough to allow access to food rewards by the forepaws, but not by the snout. (Fig. 2A). Hand-preference testing consisted of an initial training phase over the course of two weeks, followed by 50 trials per day for 4 consecutive days to determine preferential paw use. Each trial began when a 45 mg food pellet was placed into the well. Each trial ended when the rat retrieved the pellet and brought it inside the cage for consumption. All trials were recorded and reviewed using a video camera (Sony Hi-8). An observer scored the number of right and left forepaw reaches. The direction of paw preference was measured by a lateralization index computed as (L-R)/(R + L) × 100, where R and L were the number of reaches made by the right and left paws, respectively. An index of +100% and −100% corresponds to a perfect left and right-paw preference. Rats were classified as left-handed or right-handed when the lateralization index reached at least 70%. We found that about 80% of rats were right-pawed and nearly 20% of rats were left-pawed. Therefore, we retained the right-pawed rats in this study. Once paw preference was determined, we used several behavioural tests previously found to be highly sensitive to deficits in sensorimotor function (e.g., Schallert et al., 2000).

### Cylinder test

Forelimb use during spontaneous vertical exploration was analysed. The rats were placed in a transparent glass cylinder (20 cm diameter, 30 cm height) and videotaped during two sessions of 10 min each, prior to the cortical infarct, and subsequently 3 times a week after the lesion. Two mirrors positioned at a 90° angle and placed behind the glass cylinder allowed recording of forelimb movements regardless of the animal's position with respect to the camera (Fig. 2B). Slow-motion digital video playback allowed counting the number of first contacts of the paretic and non-paretic forepaw on the cylinder wall during full rear movements. An AI was computed for each testing day using the following formula: % [(paretic forelimb contacts + ½ bilateral forelimb contacts) / (paretic + non-paretic + bilateral forelimb contacts)] (Adkins et al., 2004).

The AI was averaged for each individual rat over each week examined, and a mean value was computed for all rats of the same experimental group.

### Inclined ladder test

Sensorimotor adjustments were assessed by placing the rat on a 1-meter-long ladder inclined at 45°, on which the animal had to make 5 round trips (Fig. 2C). This locomotor test allows to evaluate the sensorimotor deficits of fine sensorimotor adjustments of the forepaws on the ladder rungs. A mirror was placed under the ladder to observe the paw placement and allow for videotaping the forelimb movement and positioning on the rungs disposed at equal distances from each other. A quantitative assessment of paw placement errors was performed during slow-motion video playback. A score from 0 to 2 was assigned to the different paw placements (2: correct accurate paw placement on the rungs, 1: the animal misplaces the wrist on the rungs, 0: the paw slipped between rungs) (Martinez et al., 2009). For each forelimb and testing session, the average score was calculated as a weighted mean: [(2 × number of scores of 2) + (1 × number of scores of 1) + (0 × number of scores of 0)] / total number of scores. An asymmetry index (AI) was then calculated by comparing the scores for paretic and non-paretic forelimbs: AI = % [average score (paretic) – average score (non-paretic)] / [average score (paretic) + average score (non-paretic)]. An AI less than 0 indicated stronger functional deficits of the forelimb contralateral to the cortical lesion. On each testing day, a mean score was calculated for each paw over the 5 ascents and descents performed by the animal on the ladder. AI values were averaged weekly for each rat, and group means were calculated across all rats within each experimental group.

### Rehabilitation procedure

After the cortical lesion, rats were randomly assigned to the rehabilitated (2-W + R) or non-rehabilitated (2-W) groups. The rehabilitation procedure was carried out three times weekly, starting on the third day post-lesion and continuing for a 2-week period. The 2-W + R rats were subjected to locomotion gently imposed by a custom-made carouse device consisting of six individual boxes, rotating at a constant speed (linear speed, 8 m/min). The animals walked on carpets of varying textures and roughness (Martinez et al., 2009). Each training period comprised four 15-minute sessions, divided into two morning and two afternoon sessions (Fig. 2D–E). Between each 15-minute walking sessions, the rats were allowed to rest for 15 min. Following the morning sessions, rats were placed in an enriched environment for two hours, before initiating the afternoon sessions. This environment was equipped with diverse objects covered with textures of various roughness, thereby providing a naturalistic tactile and proprioceptive experience during the animals’ exploratory behavior (Fig. 2F). The practical advantage of this training device was to simultaneously provide several animals with similar sensorimotor stimulation regimens while providing them with a variety of tactile stimuli.

### Histology and tissue preparation

Rats were deeply anesthetized with a mixture of ketamine/medetomidine and then perfused by intracardiac injection of 400 ml of isotonic saline (0.9 % NaCl) followed by 400 ml of freshly prepared solution (4 % paraformaldehyde and in 0.1 M phosphate buffer, pH 7.4). Then, the brains were carefully dissected out and post-fixed for 12 h at 4 °C in a 4 % paraformaldehyde solution containing 10 % sucrose in phosphate buffer. The blocks were cryoprotected by successive transfers into increasing concentrations (10 %, 20 % and 30 %) of sucrose solution in 0.1 M phosphate buffer saline for 72 h at 4 °C. For histological examination, the brains were frozen using carbon dioxide, and 40-µm-thick coronal sections centred on the cortical lesions were made using a cryostat. All histological sections were mounted on slides, then processed for Nissl stain to assess morphological alterations occurring
within the infarcted zone and to measure lesion size and location with reference to the Paxinos and Watson's stereotaxic atlas (Paxinos and Watson, 2009).

We used the tracing method, more suited than the hemisphere comparison in case of microlesions (Park et al., 2013), to analyse digitized sections and obtain a stereological estimation of tissue volume loss. The infarcted tissue was manually delineated in each section based on visual identification of the high-contrast area of the cortex, using the polygon tool in ImageJ software. The lesion volume was calculated using the formula: $LV = \sum (A_i \times t)$ where LV is the total lesion volume, $A_i$ is the lesion area on slice *i* and *t* is the slice thickness. A few slices (average: $10.4 \pm 2.6$ %) were missing. In such cases, a linear interpolation was performed to estimate the lesion area. The volume of the lesion was reconstructed using a custom ImageJ script.

## Statistical methods

Our data analysis was performed with Statistica 8.0 software package (StatSoft, Tulsa, OK). The normality of data distribution was assessed using the Kolmogorov-Smirnov test, and if the data were not normally distributed, non-parametric tests were used. In addition, Friedman ANOVA was applied to compare AI scores across the post-lesion period within the 5-W group for the cylinder test, as well as during climbing and descent of the ladder. Since no significant difference was observed between the 5-W and 8-W groups in the prelesion period, their prelesion data were pooled. The Wilcoxon Matched Pairs test was then employed to compare AI scores between Prelesion and those in the 5-W and 8-W groups. For pairwise comparisons between the 5-W and 8-W groups, the Mann-Whitney *U* test was conducted. The Wilcoxon Matched Pairs test was also applied to compare scores from the cylinder and ladder tests in the *Sham* rats. To assess the impact of the rehabilitation procedure on cylinder and ladder scores, as well as to compare lesion sizes across experimental groups, Kruskal-Wallis ANOVA was performed. This test was also used to evaluate differences in forepaw map areal extent among groups. The Mann-Whitney *U* test served to compare prelesion data with results from the 8-W group. The effect of post-lesion time on RF size and location was analyzed using a factorial ANOVA, followed by a Newman-Keuls post-hoc test. A one-way ANOVA was conducted to examine how postlesion time influenced mechanical thresholds in response to ventral skin stimulation. In the *Acute* group, paired t-tests compared RF size and mechanical thresholds at similar cortical sites before and after the lesion. Finally, the Mann-Whitney *U* test was utilized to compare map areal extents between the 2-W and 2-W + *R* groups. Results are expressed as mean ± SEM throughout the manuscript, and *p*-values below 0.05 were deemed statistically significant.

## Results

### Behavioural deficits and recovery

We assessed sensorimotor skills during spontaneous and voluntary rearing behaviour using the cylinder test which probes forepaw asymmetries during upright postural support. In addition, we used the inclined ladder test which evaluates fine sensorimotor adjustments and forepaw placement during locomotion.

### Asymmetry of forepaw postural supports on the cylinder test

The cylinder task revealed that a microinfarct in M1 induced noticeable postural impairment during rearing behaviour. Friedman ANOVA revealed an increased asymmetry across the 5-W period post-lesion [$X^2(5,12) = 20.09, p = 0.001$]. The AI measured prior to the lesion averaged $60.51 \pm 3.50 \%$, attesting for a preferential use of the forepaw whose cortical representation in M1 was subsequently targeted by the cortical injury. The postlesion AI dropped to $38.78 \pm 2.82 \%$ during week 1, reflecting increased use of the non-paretic forepaw

during upright stance. This asymmetry persisted throughout the first 5 weeks, as attested by Wilcoxon Matched Pairs test comparing prelesion and 5-W groups [$z = 2.62, p = 0.009$], despite a tendency for increased contacts by the paretic forepaw over this postlesion period (Fig. 3). The mean AI significantly improved between the final assessment score in the 5-W rats ($44.07 \pm 2.71 \%$) and the 8-W rats ($57.15 \pm 3.48 \%$) as indicated by Mann-Whitney *U* test for pairwise comparison [$U = 52.00, p = 0.009$]. In addition, comparisons of the mean scores obtained prior to the lesion and at 8-W showed no significant difference [$U = 113.50, p = 0.08$]. Overall, these findings indicate that the focal injury in M1 impaired symmetric forepaw use during upright postural support with a strong tendency to neglect the paretic forepaw. Nevertheless, a complete postural symmetry was gradually achieved within approximately 8 weeks.

<figure><img src="image_5.png" /><figcaption>Fig. 3. Behavioral assessment of sensorimotor deficits using the ladder and cylinder tests. (A) Asymmetry index (AI) reflecting preferential forelimb use during rearing behaviour in the cylinder test, measured before and after the lesion. Each dot represents the weekly mean AI value (%) based on forepaw wall contacts. (B) Asymmetry index (AI) calculated from the scoring of forepaw placement on ladder rungs for the paretic and non-paretic forepaw during climbing (green) and descent (purple). The AI values were collected from the 5-W group, except for those obtained during the 8th week (8-W group). Data are shown as mean ± SEM (shaded areas). Significant differences between prelesion and post-lesion mean values are indicated, along with differences between the 5th and 8th weeks (* p < 0.01; ** p < 0.001).</figcaption></figure>
## Forepaw adjustments on the inclined ladder

Due to the relatively small number of defective paw placements (scored 1 or 0) compared to the larger number of correct placements (scored 2), and given the algorithm used in this study (difference in scores divided by the sum of scores), the Asymmetry Index (AI) produced low values. However, this algorithm is highly sensitive for detecting forepaw placement deficits, particularly when rapid and precise sensorimotor adjustments are required. Indeed, the ladder test shows that even a small number of foot faults reflect an impaired postural control. After the microlesion in M1, paw misplacements on the ladder rods were found to be more numerous for both climbing [$X^2$ (5,14) = 17.80, p = 0.003] and descent [$X^2$ (5,14) = 33.79, p < 0.0001]. Data obtained during the first postlesion weeks showed an asymmetry in postural adjustments of the forepaws both when ascending and descending the ladder (Fig. 3B). Comparison between climbing and descent over the 5-W period, using Kruskal-Wallis ANOVA, revealed a highly significant difference [H (1) = 56.89, p < 0.0001], with a systematically greater forepaw asymmetry during the descent, indicating that more precisely controlled sensorimotor adjustments were required to prevent falls on the 45° inclined ladder. The asymmetry remained relatively stable during climbing throughout the 1-W to 5-W time windows. In fact, the AI obtained prior to the lesion was smaller (0.17 ± 0.35 %) than that recorded during the 5-W period (–1.60 ± 0.32 %) [z = 2.58, p < 0.01]. By contrast, the AI during ladder descent improved gradually over the same period, as attested by the statistically significant difference between 1-W (–5.15 ± 0.58 %) and 5-W (–3.44 ± 0.32 %) [z = 3.35, p = 0.0008]. However, AI in the 5-W group remained significantly different from the Control (–0.12 ± 0.47 %) [z = 3.36, p = 0.0008] and 8-W groups (–0.29 ± 0.58 %) [U = 31.00, p = 0.0002]. By the 8-W time point, return to control values was found for both climbing [z = 1.60, p = 0.11] and descent [z = 0.05, p = 0.96]. Overall, the rats' forepaw placement ability was more severely affected during the ladder descent, compared to climbing, and was gradually restored in 8 weeks. It appears that the temporal profile of deficits assessed during the descent on the ladder closely mirrored those observed in the cylinder test throughout the postlesion period examined.

## Potential impact of surgical procedure on behaviour

Behavioural assessment was performed in sham-operated animals, which underwent the same surgical procedure as the lesioned animals prior to, and at days 3 and 6 post-surgery. No significant differences in forepaw asymmetry were found in the cylinder test when comparing the AI values measured before the surgery (55.19 ± 2.73 %) with that recorded on day 3 (49.65 ± 7.37 %) [z = 0.36, p = 0.71] and day 6 (48.07 ± 5.85 %) [z = 1.09, p = 0.27]. Comparisons of AI values assessed during ladder climbing, before the surgical procedure (0.36 ± 0.13 %) and on day 3 (–0.34 ± 0.33 %) or day 6 post-surgery (–0.13 ± 0.58 %), showed no significant differences (z = 1.46, p = 0.14; z = 0.73, p = 0.46, respectively). Similar results were found for descent, as the asymmetry recorded before the surgical procedure (0.94 ± 0.28 %) and on day 3 (0.53 ± 0.47 %) or day 6 post-surgery (1.08 ± 0.50 %) were not significantly different (z = 0.73, p = 0.46; z = 0.73, p = 0.46, respectively). Therefore, the observed behavioural deficits described above were due to the lesion rather than the surgical procedure and anesthesia.

## Effects of rehabilitative training on behavioural recovery

We sought to determine whether our rehabilitation procedure had a beneficial impact on the functional recovery of relatively undemanding versus challenging sensorimotor tasks inherent to the cylinder versus ladder tests, respectively. We compared two groups of 14-day post-lesion animals: one without rehabilitation (2-W) and the other subjected to sequences of imposed locomotion on a carpet of various textures and exposure to an enriched environment (2-W + R) (see Methods). Gross postural symmetry in forepaw touches, as assessed using the cylinder test, showed an overall positive effect of

rehabilitation on the time course of recovery [H (1) = 5.19, p = 0.023]. This effect was found to be statistically significant from the 8th to the 12th day (p < 0.03) (Fig. 4A).

For the ladder test, the analysis indicates a substantial improvement in the recovery process in the rehabilitation group for both the climbing [H (1) = 14.26, p < 0.0002] and descent [H (1) = 63.00, p < 0.0001]. However, during climbing, improvement was not consistently observed across postlesion days, in contrast to descent (Fig. 4B–C). To compare the effect of rehabilitation on climbing versus descent, we quantified the difference in mean AI between the 2-W and 2-W + R groups at each time point, separately for each direction. Comparison of the mean AI differences between climbing and descent over the postlesion period indicates a systematically greater rehabilitative effect during descent [U = 0, p = 0.005]. Overall, following the microlesion to M1, our rehabilitation procedure had a modest effect on recovery of the simpler postural task (cylinder test), in contrast to a marked improvement in behavior requesting precisely calibrated sensorimotor adjustments (descent on ladder test).

## Evaluation of lesion size and its relationship with deficits and recovery

Schematic reconstruction and representative photomicrograph of M1 microinfarcts after lesion induction is illustrated in Fig. 5A–B. These lesions targeted the forepaw representation in the CFA area of M1 following electrophysiological identification (see Methods). Microinfarctions were confined to the forepaw representation in M1 and did not spread to the neighboring forepaw area in S1, thus avoiding additional motor impairment resulting from somatosensory loss. Lesion reconstruction revealed variability in volume and extent within each group. However, the lesion volumes did not differ significantly between experimental groups, although lesion size tended to be greater at the earlier time point in non rehabilitated rats (2-W: 0.75 ± 0.17 mm³; 2-W + R: 0.59 ± 0.06 mm³; 5-W: 0.59 ± 0.10 mm³; 8-W: 0.52 ± 0.10 mm³) [H (3) = 1.33, p = 0.72].

In order to gain insight into how lesion size may have influenced the functional deficits and recovery; we analyzed the correlation between lesion volume and AI values recorded in the 5-W rats. We used the more sensitive measure of forepaw sensorimotor adjustments, i.e., the asymmetry in forepaw faults during ladder descent. We examined this anatomical-functional relationship at the 1st, 2nd, and 5th weeks post-lesion as these time windows corresponded to peak deficits expression (1st, 2nd weeks) and partial recovery (5th week). A significant negative correlation was found: the larger the lesion volume, the greater the functional asymmetry (r = -0.58, p = 0.003) (Fig. 5C).

## Organization of forepaw somatosensory maps

The cutaneous representations of the forepaw in S1 were reconstructed based on approximately 90 recording sites per mapping session. We recorded a total number of 2029 cutaneous RFs in 48 rats across the Control, Acute, 2-W, 2-W + R, 5-W, and 8-W groups in which postlesion cortical maps, except for the acute rats, were elaborated on the last day of behavioral assessment. Following cortical ministroke induction in M1, the overall extent and somatotopic organization of the forepaw cutaneous representation in S1 did not show significant changes across the post-lesion period examined [H (5) = 7.46, p = 0.19]. However, we noted a trend toward an increase in the total forepaw map extent (Control: 2.01 ± 0.16 mm², Acute: 1.98 ± 0.20 mm², 2-W: 2.21 ± 0.16 mm², 5-W: 2.63 ± 0.27 mm² and 8-W: 2.56 ± 0.23 mm²).

We reasoned that, since the glabrous skin surfaces of the forepaw are more involved than the hairy ones in sensorimotor skills during daily exploratory, feeding, or grooming behaviours, the M1 lesion may differentially affect the corresponding S1 cortical areas over the postlesion period. The areal extent of the dorsal skin regions in the map remained relatively unchanged following the lesion, except for a significant decrease during the acute stage [H (5) = 17.63, p = 0.002]. A
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 4. Effect of rehabilitation on behavioral deficits. Asymmetry indexes (%) were measured from pre-lesion to post-lesion day 14 in 2-W rats with or without rehabilitation (R). (A) *Postural asymmetry* assessed in the cylinder test showed a significant improvement from day 8 to day 12 in the rehabilitated group (\*\* p < 0.03). (B, C) *Paw placement asymmetry* during ladder climbing (B) and descent (C) showed a modest effect of rehabilitation during climbing (\* p < 0.05; \*\* p < 0.03) and a more pronounced improvement during descent (\*\*\* p = 0.01), a task requiring finer sensorimotor adjustments compared to climbing. Data are presented as mean ± SEM (shaded areas).</figcaption></figure>

trend with continuous expansion of the ventral skin area was observed throughout the post-lesion period investigated [H (5) = 9.02, p = 0.11] (Fig. 6A). Compared to the ventral skin area in *Control* rats (1.58 ± 0.12 mm²), a significant increase was observed only in *8-W* rats (2.28 ± 0.22 mm²) [U = 9.00, p = 0.015]. Comparison between the *2-W* and the *2-W + R* groups did not show any significant differences in either ventral (1.92 ± 0.17 mm² and 2.10 ± 0.16 mm²) [U = 24.50, p = 0.46] or dorsal skin areal extent (0.29 ± 0.08 mm² and 0.28 ± 0.04 mm²) [U = 114.00, p = 0.61].

### Changes in the size of cutaneous receptive fields (RFs)

#### Ventral skin RFs

We first examined acute changes in a subset of ventral skin RFs recorded from paired cortical sites (n = 98), i.e., at similar cortical locations prior to and over the first hours after the lesion. The comparison clearly shows a marked expansion of postlesion RFs (Control group: 8.91 ± 0.32 mm², Acute group: 16.23 ± 0.82 mm²; t = 9.22, p < 0.0001). It is noteworthy that 88.4 ± 1.90 % of the paired cortical sites displayed prelesion ventral skin RFs that were included within the boundaries of their postlesion RFs. When considering all the ventral skin RFs recorded in the different experimental groups, factorial ANOVA showed a main
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Relationship between asymmetry index (AI) during ladder descent and lesion volume. (A) Image of the exposed S1 cortex illustrating the location of the ministroke within the forepaw representation zone of the caudal forelimb area (CFA) in M1. The zone of vessel occlusion observed immediately after the lesion is outlined on the cortical surface (broken line). (B) Representative Nissl-stained coronal section showing cortical damage observed 5 weeks after lesion induction. (C) AI (%) measured in the 5-W group at the 1st, 2nd, and 5th weeks post-lesion are plotted against lesion volumes assessed in the same group of rats. A significant inverse correlation was observed, indicating that larger lesion volumes were associated with increased functional asymmetry (r = -0.58, p = 0.003).</figcaption></figure>

effect of experimental group [F(5,1988) = 116.37, p < 0.0001]. Newman-Keuls post-hoc tests were used to determine differences between groups. The early expansion of glabrous RFs was corroborated on the entire dataset of pre- and acute postlesion RFs (Control group: 7.06 ± 0.18 mm², Acute group: 15.30 ± 0.51 mm², p < 0.0001) (Fig. 6B). Compared to the control group, the expansion became even more prominent in the 2-W group, where RF sizes reached 18.07 ± 0.50 mm² (p < 0.0001) These findings reveal a substantial RF enlargement shortly after the lesion, with the peak expansion observed at 14 days post-lesion. At later time points, RF sizes significantly decreased. In the 5-W (5.32 ± 0.19 mm²) and the 8-W groups (3.85 ± 0.13 mm²), RFs were smaller than those recorded in the Control group (p = 0.03 and p = 0.0004, respectively). This gradual sharpening of S1 neurons’ glabrous skin RFs over the post-lesion period reflects an improvement in spatial resolution of the somatotopic map and thus a refined cortical representation as behavioural recovery developed. No significant difference was observed

between the 5-W and 8-W groups (p = 0.16), indicating a stabilization of RF sizes during this period. Notably, the mean size of glabrous skin RFs in the 2-W + R group (4.08 ± 0.11 mm²) was smaller than in the 2-W group (18.07 ± 0.50 mm²) (p < 0.0001) and as small as those recorded in the 5-W (5.32 ± 0.19 mm²) and 8-W groups (3.85 ± 0.13 mm²) (p = 0.13 and p = 0.77, respectively) (Fig. 7A).

The time course of changes in ventral RFs highlights the temporal sequence of S1 reorganization, characterized by an early post-lesion expansion followed by a gradual RF refinement and stabilization, coinciding temporally with the restoration of pre-lesion performance. It is noteworthy that RF sharpening occurred earlier in animals that underwent the rehabilitation procedure.

### Dorsal skin RFs

The analysis of dorsal RFs showed substantial alterations following the cortical lesion. Compared to the Control group (10.45 ± 0.53 mm²),
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 6.** Post-lesion evolution of S1 map features. (A) Mean areal extent (mm²) of S1 representations for glabrous and hairy skin surfaces across experimental groups. (B) Mean RF area (mm²) for neurons responsive to tactile stimulation of the hairy dorsal and glabrous ventral forepaw skin, measured in Control and at successive time points following M1 lesion. (C) Mechanical thresholds of neuronal responses to stimulation of forepaw ventral skin obtained using von Frey monofilaments (bending pressure, g/mm²), taken as measures of neuronal response sensitivity in Control and at successive time points following M1 lesion. Red triangles represent the average threshold for each experimental group, boxplots represent first and third quartiles, and medians. Data are shown as mean ± SEM (shaded areas). In A and B statistical comparisons are shown only for differences between prelesion and postlesion time points. In C, comparisons between groups are shown as horizontal bars. For additional comparisons refer to the main text. Asterisks represent significance levels: \* p < 0.05, \*\* p < 0.01, \*\*\* p < 0.001, \*\*\*\* p < 0.0001.</figcaption></figure>

RFs showed a marked expansion within the first post-lesion hours (16.04 ± 1.04 mm², p < 0.0001), an effect that persisted in the 2-W rats (16.46 ± 0.65 mm², p < 0.0001). Subsequently, RF sizes exhibited a notable reduction compared to the 2-W period (5-W: 11.66 ± 0.67 mm², p < 0.0001; 8-W: 12.49 ± 0.75 mm², p < 0.0001). This reduction led to a gradual return to pre-lesion values (p = 0.13 and p = 0.06, respectively) indicating a stabilization of RF sizes in the later phase of the recovery process (Fig. 6B). The RF expansion observed in the 2-W rats (16.46 ± 0.65 mm²) was not found in the 2-W + R group (11.75 ± 0.85 mm²) (p < 0.0001) in which RFs were not different from control (10.45 ± 0.53 mm²) (p = 0.24).

### Comparison of ventral and dorsal skin RFs

Comparison of dorsal and ventral skin RFs revealed distinct temporal dynamics across the post-lesion period (p < 0.001). In the Control group, as classically described for somatotopic maps, dorsal RFs (10.45 ± 0.65 mm²) were significantly larger than ventral RFs (7.06 ± 0.18 mm²) (p < 0.0001). However, during the Acute phase, this difference was no longer significant (p = 0.36), due to the atypical expansion of ventral RFs immediately following the lesion, as described above. By the 2-W period, ventral RFs (18.07 ± 0.50 mm²) remained as large as dorsal RFs (16.46 ± 0.69 mm²) (p = 0.06). At the 5-W and 8-W periods, the marked shrinkage of ventral RFs relative to dorsal RFs accentuated the difference between the two RFs categories (p < 0.0001). Our results also show a strong effect of the rehabilitation procedure in reducing both glabrous and hairy skin RFs, with glabrous RFs being significantly smaller (4.08 ± 0.11 mm²) than the hairy RFs (11.75 ± 0.85 mm²) (p < 0.0001) (Fig. 7A). The pronounced modulation of RF sizes throughout the post-lesion period reveals a continuous reorganization within the somatosensory cortex, characterized by an early RF expansion, at the peak of behavioral deficits, followed by differential changes for ventral (continuous decrease to below-normal sizes) and dorsal RFs (return to normal sizes) as the functional recovery was completed.

### Neuronal sensitivity to glabrous skin stimulation

We sought to evaluate neuronal responsiveness to touch as a reliable indicator of cortical neuron excitability. We thus assessed the threshold of neuronal responses reflecting the minimum sensitivity to mechanical pressure exerted at the center of glabrous skin RFs. The thresholds recorded in all post-lesion groups were found to be significantly lower than those in the Control group, indicating a prominent increase in sensitivity to light touch. More precisely, measurements of paired cortical sites in the Acute group revealed a substantial reduction in thresholds, from 11.22 ± 0.47 g/mm² to 6.79 ± 0.34 g/mm² (p < 0.0001). A similar reduction was observed across the entire ventral RF population [F(5,1643) = 598.01, p < 0.0001], with a substantial drop in threshold between the Control (15.18 ± 0.23 g/mm²) and Acute groups (7.47 ± 0.28 g/mm²) (p < 0.0001). Compared to Acute, thresholds decreased in the 2-W group (5.72 ± 0.13 g/mm²) (p < 0.0001). Thresholds remained similar in the 2-W and 5-W groups (5.81 ± 0.13 g/mm²) (p = 0.69). A further decrease was recorded between the 5-W and 8-W groups (5.07 ± 0.08 g/mm²) (p = 0.004) (Fig. 6C). The thresholds detected in the 2-W + R rats (4.90 ± 0.09 g/mm²) were lower than those recorded in the 2-W rats (5.72 ± 0.13 g/mm²) (p = 0.001) (Fig. 7B). It is noteworthy that the 2-W + R thresholds were very similar to those recorded in the 8-W rats (5.07 ± 0.08 g/mm²) (p = 0.46) which exhibited a complete behavioural recovery of fine sensorimotor abilities.

### Neuronal sensitivity and RF size correlation

Relationship between RF size and mechanical threshold has often been described in studies on somatosensory cortical plasticity. We sought to determine whether this relationship had been modified after the M1 lesion. Indeed, post-lesion changes in neuronal thresholds and RF size followed different time courses. As reported above, the thresholds
<figure><img src="image_12.png" /><figcaption>Fig. 7. Effect of postlesion rehabilitation on S1 neuron's receptive field (RF) size and responsiveness. (A) RF sizes for ventral and dorsal skin surfaces were measured 14 days after M1 lesion in rats without rehabilitation (2-W) and with rehabilitation (2-W + R). (B) Mechanical thresholds of neuronal responses assessed with Von Frey filaments in 2-W and 2-W + R rats. Mean RF areas (mm²) or thresholds (g/mm²) ± SEM are displayed above the histograms. ****: p < 0.0001, **: p = 0.001.</figcaption></figure>

decreased sharply during the *Acute* and 2-W postlesion periods, coinciding with the expansion of RF sizes. However, while a significant reduction in RF sizes was detected during the 5-W and 8-W time windows, neuronal thresholds remained substantially reduced. We first performed a multiple regression on pre-lesion sites (n = 275). The analysis revealed a significant negative correlation (r = -0.49), indicating that larger RFs were associated with lower mechanical thresholds (p < 0.0001) (Fig. 8). We carried out a similar regression analysis for the *Acute* rats (n = 406) and found that this relationship was maintained within this group, which was characterized by a large sample of expanded RFs (r = -0.43, p < 0.0001). The RF – threshold relationship was also maintained in the 2-W group (n = 298, r = -0.34, p < 0.0001), but this relationship was no longer observed in 5-W rats, despite the reappearance of neurons with higher thresholds (n = 293) (r = -0.10, p = 0.07). It is noteworthy that, in the 8-W group (n = 207), regression analysis showed an alteration in the relationship between RF size and mechanical threshold, as attested by a modest but significant positive correlation (r = 0.18, p = 0.01). In these 8-W rats, neurons exhibited both RF shrinkage and increased sensitivity, coinciding with the complete behavioural recovery of postural asymmetries. In contrast to the 2-W group, regression analysis performed in the 2-W + R rats (n = 201) showed no significant relationship between RF size and mechanical threshold (r = 0.05, p = 0.45), likely due to the marked reduction in RF size across the entire range of neuronal sensitivity.

Together, these findings indicate that the evolution of behavioural deficits following a microlesion to the forepaw sector in CFA of M1 was linked to time-dependent changes in the relationship between RF size and neuronal sensitivity to touch in the forepaw area of the S1 cortex, ultimately resulting in a modified relationship associated with the recovery state.

## Discussion

In this study, we characterized the impact of a microinfarct in the forepaw sector of the caudal forelimb area (CFA) of M1 on both sensorimotor skills and organization of the forepaw representation in S1. We assessed the postlesion cortical map remodeling and behavioural recovery over an 8-week period. In addition, we investigated the impact of

a rehabilitative procedure implemented from day 3 to day 14 postlesion on the cortical changes and sensorimotor recovery.

### Remodeling of S1 forepaw map following ministroke to M1 parallels the recovery of sensorimotor skills

Here, we demonstrate that even a microinfarct restricted to the forepaw area of the CFA induces significant changes in the corresponding ipsilateral area in S1, accompanied by measurable forelimb asymmetries in postural motor control and skilled paw placement. This functional impairment was transient, as functional recovery developed over a two-months postlesion period. Our electrophysiological mapping indicates that the overall area of the somatosensory forepaw map was not significantly modified despite a tendency to increase during the course of behavioural recovery. However, we found a selective expansion of the subregion serving the ventral skin territories, in contrast to the unchanged dorsal skin representation. This enlargement, apparently initiated during the period when marked deficits in forelimb coordination and paw placements were observed, became significant within the 8th week, when the recovery of fine sensorimotor skills was observed. Hence, a gradual restoration of complex forelimb movements occurred in parallel with the recruitment of new neuronal populations processing cutaneous information from skin surfaces mostly engaged during sensorimotor tasks. Thus, new cutaneous RFs emerged at the boundaries of the map in cortical sectors where neurons had previously displayed non-cutaneous, presumably proprioceptive responses, similarly to what was reported in monkeys trained to touch a rotating disk (Jenkins et al., 1990) and in rats exposed to an enriched environment (Coq and Xerri, 1998) or experiencing nursing behaviour (Xerri et al. 1994; Rosselet et al., 2006). In an earlier study, we demonstrated that acquisition of tactile-motor skills in monkeys leads to a substantial expansion, in somatosensory area 3b, of the representations of digit skin surfaces engaged in manual dexterity training (Xerri et al., 1996; Xerri, 1999). Following this initial study, numerous investigations in animals and humans have documented the experience-dependent plasticity of somatosensory cortical maps (for a historical overview, see Xerri, 2012). A common finding across these studies, is that the experience-induced cortical map enlargement selectively affects the sectors representing
<figure><img src="image_13.png" /><figcaption>**Fig. 8.** Changes in correlation between RF size and responsiveness for S1 neurons following M1 injury. RF sizes are plotted against thresholds of neuronal responses in all experimental groups. Note drastic alterations of the relationship between neuronal RF size and responsiveness along with the lack of return to prelesion pattern at 8 weeks postlesion, i.e., when behavioral recovery was complete.</figcaption></figure>

heavily stimulated skin territories, primarily through attention-driven behaviour. It is generally accepted that the representational expansion of glabrous skin more prominently engaged in specific behaviour reflects orderly shifts of RFs onto adjacent skin territories. The S1 neurons within the reorganized area acquire new skin locations and can be driven by a new somatosensory modality, as cutaneous inputs come to dominate proprioceptive ones, likely through a use-dependent process. More recent research has advanced our understanding of the molecular and cellular mechanisms underlying cortical plasticity driven by somatosensory experience (Kole et al., 2018; Pages et al., 2021).

Another line of research on poststroke cortical plasticity in relation to behavioural recovery has shed light on adaptive cortical changes. Notably, we found that following focal ischemic damage targeting the cutaneous representations of two digits in area 3b of S1 resulted in severe impairment of manual dexterity in monkeys. A postlesion emergence of the representation of the fingertip skin surfaces specifically involved in the digital dexterity task occurred within novel locations in area 3b in animals whose manual skills were fully restored (Xerri et al., 1998). In addition, new cutaneous representation zones for the affected digits were found to emerge in the adjacent proprioceptive cortical fields (area 1 and 3a). Thus, the recovery of manual dexterity appeared to correlate with extensive S1 remapping involving both partial restitution and substitution of the lost territories of cortical representation. Along the same lines, microlesions targeted to mouse forelimb

sensorimotor cortex were found to induce a sensory remapping into the peri-infarct motor or more distant regions (Brown et al., 2009; Harrison et al., 2013), a change closely related to increased spontaneous use of the paretic forelimb (Brown et al., 2009). The adaptive S1 remodelling reported herein may also, at least in part, be ascribed to the recruitment of the rostral forelimb area (RFA), primarily devoted to grasping (Brown and Teskey, 2014) and considered a rodent homologue of the premotor cortex that normally receives forelimb-related sensory inputs from the CFA via the cortico-cortical pathway (Kunori and Takashima, 2016). In fact, four weeks after an ischemic lesion to the CFA, increased connectivity between the RFA and S1, along with reinforced modulation of sensory responses in S1 by premotor activity, has been disclosed during forelimb skills recovery, suggesting a newly organized sensorimotor integration after injury (Hayley et al., 2023). Approximately 12 weeks after a ministroke in the M1 hand representation of squirrel monkeys, an enlarged hand representation was found in the ipsilateral ventral premotor cortex (PMV) (Frost et al., 2003), a region representing a major source of intracortical inputs to M1 and known to be involved in the spatial guidance of limb movements (Kakei et al., 2001). Interestingly, this enlargement was proportional to the amount of hand representation destroyed in M1, suggesting that post-stroke cortical plasticity may be driven by reciprocal intracortical connections between the damaged and intact areas. To our knowledge, the present study is the first to show that a focal injury to the forepaw area in M1 leads to a selective, presumably
compensatory, expansion of the ipsilesional representation of its somatosensory counterpart in S1, thus providing further evidence for the tight functional links between both cortical areas. Hypothetically, following M1 injury, due to the impairment of forepaw motor commands, the continued expansion of the corresponding cutaneous area in S1 may, at least in part, express a gradual sensorimotor recalibration between actual and expected movements. Such a recalibration process would be critically important for complex sensorimotor adjustments requiring increased reliance on sensory feedback and enhanced attentional engagement to successfully adapt motor planning and execution to challenging sensorimotor tasks. Increased attention to peripheral somatosensory inputs is a prerequisite for the cortical reorganization to underlie fine sensorimotor skills, as previously demonstrated (Recanzone et al. 1992; Xerri et al., 2005). It is noteworthy that the forepaw area enlargement described herein was observed in spontaneously recovering animals, as was the above-mentioned postinfarct increase in the paretic hand representation in PMV (Frost et al., 2003). This motor map enlargement occurred in parallel with a substantial shrinkage of digit representations in the perilesional M1 cortex (Nudo and Milliken, 1996). Interestingly, the enlarged forelimb representation in PMV was accompanied by a proliferation over a five-month period of novel terminal fields from PMV to the areas 1 and 2 of S1 (Dancause et al., 2005). This increased PMV-S1 connectivity likely contributed to compensating for the stroke-induced loss of anatomical projections from M1 to S1, thereby facilitating the recovery of manual skills. However, a direct link between reorganization of PMV and recovery of manual skills was not established in that study. The present findings support the view that M1 and S1 are tightly interconnected through corticocortical and thalamocortical loops to form an integrated sensorimotor network crucial for movement generation. In this dynamic network, M1 continuously integrates sensory input to update and shape motor output (Nudo, 2007; Brown et al., 2009) while exerting an ongoing influence on the functional properties of S1 (for review, Borich et al., 2015).

It is generally admitted that cortical map expansion results from changes in the balance of excitation and inhibition within thalamocortical and intracortical connectivity through unmasking or strengthening of pre-existing connections, so that stronger inputs become effective in exciting larger populations of neurons (for reviews, see Buonomano and Merzenich, 1998; Jones, 2000; Xerri et al., 2014). It is worth mentioning that long-term potentiation (LTP) was found to occur in S1 following M1 stimulation in cats (Keller et al., 1990) indicating that M1 activity plays an active role in use-dependent somatosensory cortex plasticity. Particularly relevant to the cutaneous map expansion observed in the present study is the finding that neurons initially responsive only to high-threshold inputs—such as light tapping of the forepaw, which elicits non-cutaneous responses—began firing in response to light cutaneous stimulation following ACh application (Metherate et al., 1988). Furthermore, the basal forebrain cholinergic system has been identified as a critical mediator of cortical plasticity, particularly in supporting the expansion of motor representations in perilesional areas, which underlies the recovery of skilled reaching after focal injury to the motor cortex (Conner et al., 2005). The possibility of structural changes should not be overlooked when explaining cortical map remodelling. In this regard, it has been proposed that early synchronous neuronal activity recorded in the perilesional cortex following thermal-ischemic injury is an initial trigger for axonal sprouting facilitating synaptic remodelling and long-term cortical reorganization (Carmichael and Chesselet, 2002).

## Postlesion changes in S1 neurons' RFs temporally coincide with the functional deficits and subsequent recovery

In contrast to the relatively continuous postlesion expansion of the ventral skin area in the S1 forepaw map, constituent neurons displayed RFs and sensitivity to touch that evolved with distinct temporal dynamics. More precisely, changes in RFs' sizes were characterized by an

immediate post-lesion enlargement reaching a peak during the second postlesion week, i.e., during the acute and subacute phases of M1 infarction. This expansion was followed by a gradual contraction leading to smaller-than-normal ventral skin RFs, while dorsal skin RFs returned to prelesion sizes. The initial RF expansion very likely impaired tactile acuity by degrading the spatial resolution of the somatosensory map. This prominent RF alteration which occurred over a period of greater behavioural deficits may have aggravated the deleterious effects of the M1 lesion on corticospinal output. In our view, the rapid, undifferentiated enlargement of both ventral and dorsal skin RFs testifies for reactive plasticity triggered by disruption of the normal state of M1-S1 interactions. In rodents, M1 and S1 form reciprocal excitatory and inhibitory connections (Mao et al., 2011; Kinnischtzke et al., 2014), with M1 modulating S1 network dynamics and thereby improving sensory discrimination during tactile exploration (Zagha et al., 2013). It is noteworthy that, following focal ischemic stroke in the mouse vibrissa representation in M1, the loss of M1-to-S1 inhibition impaired the reliability of responses to whisker stimulation during the acute phase (first week postlesion) (Fukui et al., 2020). It has long been argued that the balance of excitatory and inhibitory influences critically modifies cortical neurons' properties (RF location, size and responsiveness) by modulating the synaptic strength of subthreshold and suprathreshold inputs ultimately converging onto cortical cells (Calford, 2002; Jones, 1993; Xerri, 2012). A widespread reduction of GABA<sub>A</sub>-receptor expression in the perilesional M1 cortex has brought evidence for a strong intracortical disinhibition (Schiene et al., 1996). Previous studies have demonstrated that pharmacological reduction of GABAergic inhibition within S1 leads to an immediate enlargement of cutaneous RFs (Alloway et al., 1989; Chowdhury and Rasmusson, 2002; Dykes et al., 1984; Tremere et al., 2001). This decrease in cortico-cortical inhibition is consistent with our findings showing a rapidly initiated enhancement of neuronal excitability, as indicated by a marked reduction in the response threshold of S1 neurons to light touch. This finding is in line with previous observations showing that, within 50 min of a focal ischemic injury to M1, S1 neuronal responses to median nerve stimulation were substantially enhanced, resulting in expansion of the responsive cortical area, reduction of response latency, prolonged activation and increased response amplitude, all effects attributed to a loss of intracortical inhibition (Paul et al., 2006). This early release of inhibition leading to enhanced effectiveness of broad sets of convergent cutaneous inputs may have promoted the unmasking of previously latent intracortical and thalamocortical synapses, i.e., synapses that had not previously contributed to neuronal discharge (Jacobs and Donoghue, 1991; Malenka and Nicoll, 1997). As a result, effective inputs from nearby skin regions onto S1 neurons may have caused increased excitability and synaptic remodelling leading to RF expansion. The contribution of NMDA to immediate RF changes seems unlikely, as NMDA receptors upregulation has been shown to occur with a delay of about 2 weeks after focal cortical injury (Que et al., 1999). In addition, the possibility cannot be excluded that early disuse of the paretic limb, attested herein during the animal upright stance, led to reduced thalamocortical inhibition, which may have contributed to the cutaneous RFs' enlargement, as previously observed following forelimb immobilization or exposure to environmental impoverishment (Coq and Xerri, 1999).

In the present study, we provide evidence for a gradual sharpening of both ventral and dorsal skin RFs as behavioural recovery progressed. The observation that the RF contraction was pre-eminent for ventral skin surfaces at the time of behavioural recovery is consistent with the more salient involvement of the ventral skin in adjusting forepaw placements. In line with previous studies, it is likely that the gradual reduction in RF size leading to a finer spatial resolution of the somatopic map concomitantly with the selective expansion of the glabrous skin representation, contributed to enhanced tactile acuity (Jenkins and Merzenich, 1987; Bourgeon et al., 2004; Xerri et al., 2005; Rosselet et al., 2006). The functional significance of sensory signals from S1 to M1 is thought to be related to sensorimotor integration and motor skill
learning (Sakamoto et al., 1987; Pavlides et al., 1993; Xu et al., 2012). We mentioned above that M1 focal infarction impaired the spatiotemporal dynamics of sensory processing in S1 during the acute phase of the lesion through a release of inhibition from M1 to S1. (Fukui et al., 2020). The disruption of temporal coding in S1 induced a sensory deficit which was followed by recovery during the subacute phase of infarction (second week), with the late component of the response to whisker deflection being increased. This late component of stimulus-evoked neuronal activity in S1 is believed to involve top-down control and correlate with conscious perception of somatosensory stimuli (Del Cul et al., 2007; Manita et al., 2015). Taken together, these findings suggest that functional recovery may be considered a compensatory attention-driven relearning process in which improvements in sensorimotor performance depend on error-driven changes within interactive somatosensory and motor networks, occurring in a behavioural context of heightened attention due to a mismatch between task demands and actual sensorimotor capabilities.

In our view, unlike the immediate RF expansion reflecting *reactive plasticity* primarily mediated by cortico-cortical interactions, RF sharpening represents *adaptive plasticity* driven by experience-dependent mechanisms. There is ample evidence that cortical maps are continuously reorganized and refined through activity-dependent adjustments in the balance of inhibitory and excitatory intracortical and thalamocortical influences (for reviews, see Buonomano and Merzenich, 1998; Caleo, 2015). The study of experience-dependent changes in the S1 ventrum representation during lactation-related nursing revealed that GAD65 and GAD67 expression was upregulated at a time when RFs were smaller and subsequently downregulated to pre-nursing levels as RF sizes returned to baseline (Rosselet et al., 2006). Cortical neuron RFs were reshaped on a timescale closely linked to nursing-driven changes in somatosensory inputs. This finding is in agreement with the available evidence from other studies suggesting that the RF shrinkage may be ascribed to increased levels of GAD, very likely through use-dependent and selective suppression of suprathreshold excitatory inputs (Dykes et al., 1984; Foeller et al., 2005; Gierdalski et al., 2001; Tremere et al., 2001; Welker et al., 1989) and the formation of GABAergic synapses (Knott et al., 2002). In the present study, the RF sharpening, ascribed to a use-dependent enhancement of inhibition, is not inconsistent with the observed increase in neuronal responsiveness. In effect, it is likely that subsets of excitatory synapses, among converging thalamocortical and intracortical connections, may have been strengthened through behaviorally-driven Hebbian-like synaptic plasticity, a mechanism showed to only operate with partial blockage of cortical GABA<sub>A</sub> receptors (Castro-Alamancos and Borrel, 1995). Although the current discussion emphasizes the role of GABAergic mechanisms in cortical plasticity, the contribution of acetylcholine (ACh) should not be overlooked. Notably, activation of cholinergic pathways in the basal forebrain has been shown to induce long-term changes in the excitability of neurons in the somatosensory cortex, characterized by an increased response magnitude and a lowered threshold to cutaneous stimuli (Metherate et al., 1988; Tremblay et al., 1990). Also, ACh liberation contributes to a long-lasting strengthening of neuronal connections (Verdier and Dykes, 2001).

## Effect of a naturalistic rehabilitative procedure on cortical remodelling and recovery of fine sensorimotor skills

Our rehabilitation paradigm, combining walking on textured surfaces of varying rugosity with exposure to an enriched environment (EE), was found to be effective in promoting faster recovery of forepaw postural symmetry and fine sensorimotor adjustments. The time gain in recovery was mainly observed in the task requiring precisely recalibrated sensorimotor adjustments (ladder descent). Furthermore, it was paralleled with refinement of S1 neurons’ glabrous skin RFs and increased sensitivity to light touch, very similar to that observed in the recovery state of 8-W non-rehabilitated animals. Surprisingly, milder

deficits on the ladder descent were found to occur as early as the 4th day postlesion in the rehabilitated rats. Several non-mutually exclusive mechanisms, such as early changes within the perilesional M1 cortex and altered S1–M1 interactions, may underlie the rapid behavioural improvements observed with rehabilitation training. *First*, we used microinfarcts significantly smaller than those typically employed in rat models (e.g., 5–7 mm³), which may have enabled quicker neuroprotective and plastic responses. Because ischemic damage often spreads beyond the lesion core, smaller lesions might limit secondary damage and favor early repair in perilesional areas. *Second*, our data show that rehabilitation induced neuronal changes in S1, specifically, increased responsiveness and refined cutaneous receptive fields, features linked to improved tactile discrimination, perceptual learning and manual skills. These adaptive changes likely began before the second week post-lesion. S1 and M1 are interconnected via direct corticocortical projections, especially from S1 layers 2/3 and 5A to M1 (Yamawaki et al., 2021), providing a structural basis for S1 activity to influence M1. Furthermore, S1 hyperexcitability can emerge shortly after M1 injury. For instance, Paul et al. (2006) reported heightened and faster somatosensory responses within the first hour following a photothrombotic M1 lesion. These rapid changes in S1 may support early compensatory activation of perilesional M1, mitigating deficits and potentially facilitating recovery through activity-dependent plasticity. Most studies on post-stroke rehabilitation have focused on early (1–7 days) or subacute (7 days–3 months) phases, while ultra-early interventions (≤24 h) have often been avoided due to possible adverse effects. This may have led to an underestimation of their potential benefits. Notably, in stroke patients, even a single session of constraint-induced movement therapy (CIMT) has been shown to enhance motor output to paretic muscles and manual dexterity within 24 h, as measured by TMS over M1 (Liepert et al., 2000). The expanded cortical representation of paretic muscles suggests recruitment of a broader neuronal pool, possibly through disinhibition or synaptic strengthening (Iriki et al., 1989; Jacobs and Donoghue, 1991), facilitating perilesional reactivation. Similarly, Geng et al. (2022) showed that initiating rehabilitation 24 h after MCAO in rats led to significant functional recovery by day 3, along with reduced neuronal apoptosis, increased neuronal survival, and angiogenesis. In the same study, stroke patients who began rehabilitation at 24 h showed greater improvements in motor and daily living functions at 1 and 3 months compared to those who started at 72 h, supporting the efficacy of ultra-early rehabilitation. In addition, previous studies have demonstrated that EE housing (Kieling et al., 2009; Balbinot et al., 2023) or rehabilitative training (Nudo et al., 1996; Nishibe et al., 2015; Wang et al., 2016; Mascaro et al., 2019; Conti et al., 2022) enhances cortical plasticity and recovery of skilled reaching following focal M1 infarction. For example, a 20-minute tray-reaching task that required global grasping movements from post-lesion days 8 to 12, led to a more rapid improvement in motor performance and movement quality on a skilled reaching task during the training period, which persisted throughout the subsequent three weeks (Nishibe et al., 2015). The authors showed that approximately five weeks after the lesion, forelimb motor maps in the rehabilitative training group of rats were significantly enlarged compared to the non-rehabilitated group. This finding corroborates an earlier study revealing that, in contrast to untrained monkeys, those that benefited from a rehabilitative training involving the paretic hand had preserved or even enlarged sectors of the hand motor representation (Nudo and Milliken, 1996). Several studies have shown that the timing and intensity of rehabilitative intervention are critical factors in promoting functional recovery. For example, overuse of the paretic limb by immobilizing the intact forelimb during the first two weeks post-stroke was found to impede motor recovery and increase lesion volume in rats (Kozlowski et al., 1996; Humm et al., 1998; Bland et al., 2000). By contrast, a model of constraint-induced movement therapy (CIMT) showed that a moderate-intensity treatment (8 h/day), initiated immediately after focal cortical ischemia, promoted the recovery of forelimb skills and symmetrical use (Joo et al., 2012). A study combining EE and
daily food-pellet retrieval in a reaching task, initiated at 5, 14 or 30 days after middle cerebral artery occlusion, showed that functional restoration of skilled forelimb reaching and ladder-rung-walking was most effective with the earliest rehabilitation protocol (Biernaskie et al., 2004). We have previously shown that early cutaneous stimulation of the forepaw, delivered in short daily sessions during the first post-lesion week, in an attention-demanding behavioural context, was sufficient to limit cortical tissue loss and exert a protective effect on *peri-infarct zones* of the forepaw map specifically serving the stimulated skin surfaces (Xerri and Zennou-Azogui, 2014). As previously noted, the basal forebrain plays a crucial role in experience-dependent cortical plasticity. Cortical acetylcholine release has been shown to correlate with exploratory behaviour in response to environmental novelty (Acquas et al., 1996). Moreover, expansion of the RFA following focal cortical injury in the CFA was found to be dependent on cholinergic activity (Conner et al., 2005). Importantly, this expansion that was essential for functional recovery occurred only when rehabilitative training was implemented. Consistently, cholinergic depletion after CFA lesions significantly reduced rehabilitation-driven dendritic complexity and spine number in the perilesion cortex and markedly attenuated functional recovery (Wang et al., 2016). Collectively, these findings suggest that our rehabilitation protocol, which combines environmental enrichment with sensorimotor stimulation, likely engages cholinergic mechanisms critical for promoting cortical plasticity. Overall, these studies plead in favor of an early critical period characterized by heightened sensitivity to rehabilitative experience and of particular vulnerability to the deleterious effects of intensive training. Our findings indicate that early, yet moderate, rehabilitative training promotes adaptive cortical remodelling and recovery of fine sensorimotor skills following a ministroke in M1. Assuming that the rehabilitative procedure used in the present study had neuroprotective effects on *peri*-lesional tissue in M1 or even promoted the emergence of new sectors of forelimb representation in this cortical area (Mascaro et al., 2019), these beneficial effects did not prevent concurrent changes in S1 forepaw area. In the present study, we did not assess whether the alterations in S1 neuron properties—namely, a larger ventral forepaw representation, smaller ventral skin receptive fields (RFs), and greater sensitivity—were transitory or permanent, as we did not examine the post-lesion period beyond the eighth post-lesion week. Our findings suggest that the recovery of sensorimotor skills following focal M1 injury may not reflect a restitution of the pre-lesion state of interactions within the M1-S1 network. Instead, they indicate the emergence of newly organized interactions that result in a sustained increase in reliance on somatosensory feedback for the control of fine sensorimotor skills. Future longitudinal studies could explore how neuronal responses in S1 change following lesions of varying sizes and locations within the CFA and RFA. Incorporating post-injury ICMS remapping could help track alterations in these motor areas. However, conducting cortical mapping and remapping experiments in the same animals remains a significant challenge. Furthermore, the postlesion temporal dissociation observed between the RF size and neuronal responsiveness to tactile input calls for studies aimed at characterizing the synaptic, cellular, or circuit-level processes underlying this divergence, which may hold key implications for understanding sensory-driven recovery.

### CRediT authorship contribution statement

**Fatemehsadat Abtahi:** Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation, Formal analysis, Data curation. **Yoh'i Zennou-Azogui:** Writing – review & editing, Visualization, Validation, Investigation, Formal analysis, Data curation. **Sabine Quitard:** Software. **Christian Xerri:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

This research was supported by grants from the Ministère de l'Enseignement Supérieur et de la Recherche and the CNRS (Aix-Marseille Université, AMU, UMR 7077). A fellowship grant from the AMU NeuroSchool was provided by the Agence Nationale de la Recherche (ANR).

We thank Elodie Mansour and Jean Luc Fina for taking care of the animals, and Isabelle Watabe for her helpful assistance during the historical procedure.

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