{"paper_id":"018f06a8-149a-4d1e-9432-1a4bc68bacfd","body_text":"Title: Changes in intra- and interlimb reflexes from forelimb cutaneous afferents after staggered thoracic 1 \nlateral hemisections during locomotion in cats 2 \nRunning title: Reflexes in the four limbs during locomotion after spinal cord injury 3 \nAuthor names: Stephen Mari1, Charly G. Lecomte1, Angèle N. Merlet1, Johannie Audet1, Sirine Yassine1, 4 \nRasha Al Arab1, Jonathan Harnie1, Ilya A. Rybak2, Boris I. Prilutsky3 and Alain Frigon1 5 \n 6 \nAffiliations: 1Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université 7 \nde Sherbrooke, Centre de recherche du Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, QC, 8 \nCanada; 2Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, 9 \nPA, United States; 3School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, United 10 \nStates 11 \nKeywords: cutaneous reflexes, interlimb coordination, locomotion, spinal cord injury 12 \nSupport or grant information: This work was supported by a grant from the National Institutes of Health: 13 \nR01 NS110550 to AF, IAR and BIP. AF is a Fonds de Recherche-Santé Quebec (FRQS) Senior Research 14 \nScholar. JA and JH were supported by FRQS doctoral scholarships and ANM by a FRQS postdoctoral 15 \nscholarship. 16 \n 17 \nAuthor contributions: SM, IAR, BIP, and AF contributed to conception and design of the study. SM, CL, 18 \nAM, JA, SY, RA and JH conducted the research. SM organized the database and performed the data and 19 \nstatistical analysis. SM and AF wrote the first draft of the manuscript. All authors contributed to manuscript 20 \nrevision, read, and approved the final version. 21 \n 22 \nAcknowledgments: We thank Philippe Drapeau for providing data acquisition and analysis software, 23 \ndeveloped in the Rossignol and Drew laboratories at the Université de Montréal. We thank the Biostatistics 24 \ndepartment of the Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke for statistical 25 \nassistance.  26 \n 27 \nData availability statement: The raw data supporting the conclusions of this article will be made available 28 \nby the authors, without undue reservation.  29 \n 30 \nEthics statement: The animal study was reviewed and approved by the Animal Care Committee of the 31 \nUniversité de Sherbrooke. 32 \n 33 \nCorresponding author: 34 \nAlain Frigon, PhD 35 \nEmail: alain.frigon@usherbrooke.ca 36 \nORCID ID: 0000-0002-9259-2706 37 \nCompeting statement: The authors declare no competing financial interests. 38 \n 39 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nAbstract:  40 \nIn quadrupeds, such as cats, cutaneous afferents from the forepaw dorsum signal external perturbations and 41 \nsend signals to spinal circuits to coordinate the activity in muscles of all four limbs. How these cutaneous 42 \nreflex pathways from forelimb afferents are reorganized after an incomplete spinal cord injury is not clear. 43 \nUsing a staggered thoracic lateral hemisections paradigm, we investigated changes in intralimb and interlimb 44 \nreflex pathways by electrically stimulating the left and right superficial radial nerves in seven adult cats and 45 \nrecording reflex responses in five forelimb and ten hindlimb muscles. After the first (right T5-T6) and second 46 \n(left T10-T11) hemisections, forelimb-hindlimb coordination was altered and weakened. After the second 47 \nhemisection, cats required balance assistance to perform quadrupedal locomotion. Short-, mid- and long-48 \nlatency homonymous and crossed reflex responses in forelimb muscles and their phase modulation 49 \nremained largely unaffected after staggered hemisections. The occurrence of homolateral and diagonal mid- 50 \nand long-latency responses in hindlimb muscles evoked with left and right superficial radial nerve stimulation 51 \nwas significantly reduced at the first time point after the first hemisection, but partially recovered at the 52 \nsecond time point with left superficial radial nerve stimulation. These responses were lost or reduced after 53 \nthe second hemisection. When present, all reflex responses, including homolateral and diagonal, maintained 54 \ntheir phase-dependent modulation. Therefore, our results show a considerable loss in cutaneous reflex 55 \ntransmission from cervical to lumbar levels after incomplete spinal cord injury,  albeit with preservation of 56 \nphase modulation, likely affecting functional responses to external perturbations.  57 \n 58 \nKey points:  59 \n• Cutaneous afferent inputs coordinate muscle activity in the four limbs during locomotion when the forepaw 60 \ndorsum contacts an obstacle.  61 \n• Thoracic spinal cord injury disrupts communication between spinal locomotor centers located at cervical 62 \nand lumbar levels, impairing balance and limb coordination. 63 \n• We investigated cutaneous reflexes from forelimb afferents during quadrupedal locomotion by electrically 64 \nstimulating the superficial radial nerve bilaterally, before and after staggered lateral thoracic hemisections in 65 \ncats. 66 \n• We showed a loss/reduction of mid- and long-latency homolateral and diagonal reflex responses in 67 \nhindlimb muscles early after the first hemisection that partially recovered with left superficial radial nerve 68 \nstimulation, before being reduced after the second hemisection. 69 \n• Targeting cutaneous reflex pathways from forelimb afferents projecting to the four limbs could help develop 70 \ntherapeutic approaches aimed at restoring transmission in ascending and descending spinal pathways.  71 \n 72 \n 73 \n 74 \n 75 \n 76 \n 77 \n 78 \n 79 \n 80 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nINTRODUCTION 81 \n locomotion, inputs from the skin provide information on the external environment, such as characteristics 82 \nof the terrain and obstacles encountered (Rossignol et al., 2006; Pearcey & Zehr, 2019a; Frigon et al., 83 \n2021). For instance, in cats and humans, cutaneous afferents play an important role in modifying limb 84 \ntrajectory and maintaining balance during locomotion when the foot/hindpaw dorsum , innervated by the 85 \nsuperficial peroneal (SP) nerve, contacts an obstacle during the swing phase, termed the stumbling 86 \ncorrective reaction (Forssberg et al., 1977; Prochazka et al., 1978; Forssberg, 1979; Duysens & Loeb, 1980; 87 \nWand et al., 1980; Schillings et al., 1996; Van Wezel et al., 1997; Zehr et al., 1997; Quevedo et al., 2005b, 88 \n2005a). In quadrupeds, such as cats, stimulating the dorsum of the forepaw or the superficial radial ( SR) 89 \nnerve also elicits a stumbling corrective reaction that alters forelimb trajectory  to move it away and over a 90 \nsimulated obstacle (Miller et al., 1977; Matsukawa et al., 1982; Drew & Rossignol, 1985, 1987; Shimamura 91 \net al., 1990; Fuwa et al., 1991; Hurteau et al., 2018; Mari et al., 2023). In cats and humans, electrically 92 \nstimulating the SP and SR nerves evoke short-, mid- and long-latency inhibitory and/or excitatory cutaneous 93 \nreflex responses in muscles of the four limbs that are modulated with task and phase during locomotion 94 \n(Haridas & Zehr, 2003; Mari et al., 2023). Mid- and long-latency responses are thought to involve supraspinal 95 \ncontributions, but also polysynaptic spinal pathways (Fuwa et al., 1991; LaBella et al., 1992; Pijnappels et 96 \nal., 1998; Christensen et al., 1999; Hiersemenzel et al., 2000; Frigon & Rossignol, 2008; Hurteau & Frigon, 97 \n2018; Duysens 2024). 98 \nSpinal cord injury (SCI) disrupts ascending and descending pathways that communicate between the 99 \nbrain and spinal networks controlling arm/forelimb and leg/hindlimb movements, including those activated 100 \nand modulated by cutaneous inputs. The disruption of these pathways and associated sensorimotor deficits 101 \nin balance and limb coordination vary depending on the severity and level of the lesion (Barbeau et al., 2002; 102 \nEdgerton et al., 2004; Frigon & Rossignol, 2006; Rossignol & Frigon, 2011). To investigate how SCI affects 103 \ncutaneous reflex pathways projecting to the four limbs, we recently used a staggered thoracic lateral 104 \nhemisections paradigm in cats and evoked cutaneous reflex responses by stimulating the SP nerve (Mari et 105 \nal., 2024). Staggered thoracic hemisections disrupt direct communication between the brain/cervical cord 106 \nand lumbar locomotor networks, disrupting fore-hind coordination and balance in cats and rats (Jane et al., 107 \n1964; Kato et al., 1984, 1985; Stelzner & Cullen, 1991; Courtine et al., 2008; Van Den Brand et al., 2012; 108 \nCowley et al., 2015; Audet et al., 2023). We reported a significant reduction in the presence of mid- (19-34 109 \nms) and long-latency (35-60 ms) responses in muscles of all four limbs, especially in the forelimbs (Mari et 110 \nal., 2024). In humans, a few studies have shown changes in interlimb reflexes (i.e. from the arms to the legs 111 \nor from the legs to the arms) after spinal cord injury, but only at rest (Calancie, 1991; Calancie et al., 1996, 112 \n2002; Butler et al., 2016). Thus, we do not know how reflexes evoked by forelimb afferents are reorganized 113 \nafter SCI during locomotion.  114 \nTherefore, the purpose of the present study was to investigate reflex responses evoked by stimulating the 115 \nSR nerve before and after staggered thoracic hemisections in the same animal during treadmill locomotion, 116 \nextending our previous findings with responses evoked by hindlimb cutaneous afferents (Mari et al., 2024). 117 \nWe first assessed SR-evoked reflex responses in the intact state and then following a first lateral 118 \nhemisection at mid-thoracic (T5-T6) on the right side. We then assessed how SR-evoked reflex responses 119 \nchanged following a second lateral hemisection at left T10-T11. The main finding was a loss/reduction in 120 \nhomolateral and diagonal responses in hindlimb muscles after staggered thoracic hemisections, which 121 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\ncorrelated with weakened coordination between the fore- and hindlimbs and impaired balance during 122 \nquadrupedal locomotion (Audet et al., 2023). 123 \n 124 \nMATERIAL AND METHODS 125 \nEthical approval  126 \nAll procedures were approved by the Animal Care Committee of the Université de Sherbrooke (Protocol 127 \n442-18) in accordance with policies and directives of the Canadian Council on Animal Care. We obtained the 128 \ncurrent data set from seven adult purpose-bred cats (> 1 year of age at the time of experimentation), 3 129 \nfemales and 4 males, weighing between 3.4 kg and 6.5 kg, purchased from Marshall BioResources  (North 130 \nRose, NY, USA). Before and after experiments, cats were housed and fed (weight-dependent metabolic diet 131 \nand water ad libitum) in a dedicated room within the animal care facility of the Faculty of Medicine and Health 132 \nSciences at the Université de Sherbrooke. We followed the ARRIVE guidelines 2.0 for animal studies 133 \n(Grundy, 2015; Percie Du Sert et al., 2020). The investigators understand the ethical principles under which 134 \nthe journal operates and our work complies with this animal ethics checklist. In order to maximize the 135 \nscientific output of each animal, they were used in other studies to investigate different scientific que stions, 136 \nsome of which have been published (Lecomte et al., 2022, 2023; Merlet et al., 2022; Audet et al., 2023; Mari 137 \net al., 2023, 2024).  138 \n 139 \nGeneral surgical procedures 140 \nAll surgeries (implantation and spinal lesions) were performed under aseptic conditions with sterilized 141 \nequipment in an operating room. Prior to surgery, cats were sedated with an intramuscular (i.m.) injection of 142 \nbutorphanol (0.4 mg/kg), acepromazine (0.1 mg/kg), and glycopyrrolate (0.01 mg/kg). We th en injected a 143 \nmixture (0.05 ml/kg, i.m.) of diazepam (0.25 mg/kg) and ketamine (2.0 mg/kg) in a 1:1 ratio five minutes later 144 \nfor induction. We shaved the animal’s fur (back, stomach, fore- and hindlimbs) and cleaned the skin with 145 \nchlorhexidine soap. Cats were anesthetized with isoflurane (1.5-3%) and O2 delivered with a mask and then 146 \nwith a flexible endotracheal tube. The depth of anesthesia was confirmed by applying pressure to a paw (to 147 \ndetect limb withdrawal) and by assessing the size and reactivity of pupils. Isoflurane concentration was 148 \nadjusted throughout the surgery by monitoring cardiac and respiratory rates . Body temperature was 149 \nmaintained constant (37 ± 0.5°C) using a water-filled heating pad placed under the animal, an infrared lamp 150 \nplaced ~50 cm over it and a continuous infusion of lactated Ringers solution (3 ml/kg/h) through a catheter 151 \nplaced in a cephalic vein. At the end of surgery, we injected subcutaneously an antibiotic (cefovecin, 8 152 \nmg/kg) and a fast-acting analgesic (buprenorphine, 0.01 mg/kg). We also taped a fentanyl (25 µg/h) patch to 153 \nthe back of the animal 2-3 cm rostral to the base of the tail for prolonged analgesia, which we removed 4-5 154 \ndays later. After surgery, cats were placed in an incubator and closely monitored until they regained 155 \nconsciousness. We administered another dose of buprenorphine ~7 hours after surgery. At the end of 156 \nexperiments, cats were anaesthetized with isoflurane (1.5–3.0%) and O2 before receiving a lethal dose of 157 \npentobarbital (120 mg/kg) through the left or right cephalic vein. Cardiac arrest was confirmed using a 158 \nstethoscope to determine the death of the animal. Spinal cords were then harvested for histological analysis 159 \n(Lecomte et al., 2022, 2023; Audet et al., 2023; Mari et al., 2024). 160 \n 161 \n 162 \n 163 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nStaggered lateral hemisections 164 \nAfter collecting data in the intact state, we performed a lateral hemisection between the 5th and 6th 165 \nthoracic vertebrae (T5-T6) on the right side of the spinal cord. Before surgery, we sedated the cat with an 166 \nintramuscular injection of a cocktail containing butorphanol (0.4 mg/kg), acepromazine (0.1 mg/kg) and 167 \nglycopyrrolate (0.01 mg/kg) and inducted with another intramuscular injection (0.05 ml/kg) of ketamine (2.0 168 \nmg/kg) and diazepam (0.25 mg/kg) in a 1:1 ratio. We shaved the fur overlying the back and t he skin was 169 \ncleaned with chlorhexidine soap. The cat was then anesthetized with isoflurane (1.5 -3%) and O2 using a 170 \nmask for a minimum of 5 minutes and then intubated with a flexible endotracheal tube. Isoflurane 171 \nconcentration was confirmed and adjusted throughout the surgery by monitoring cardiac and respiratory 172 \nrates, by applying pressure to the paw to detect limb withdrawal and by assessing muscle tone. Once the 173 \nanimal was deeply anesthetized, an incision of the skin over and between the 5th and 6th thoracic vertebrae 174 \n(T5-T6) was made and after carefully setting aside muscle and connective tissue, a small laminectomy of the 175 \ncorresponding dorsal bone was performed. Lidocaine (xylocaine, 2%) was applied topically followed by 2 -3 176 \nintraspinal injections on the right side of the cord. We then sectioned the spinal cord laterally from the midline 177 \nto the right using surgical scissors. We placed hemostatic material (Spongostan) within the gap before 178 \nsewing back muscles and skin in anatomical layers. In the days following hemisection, voluntary bodily 179 \nfunctions were carefully monitored. The bladder and large intestine were manually expressed if needed. 180 \nOnce data were collected following the first hemisection (9-13 weeks), we performed a second lateral 181 \nhemisection between the 10th and 11th thoracic vertebrae (T10-T11) on the left side of the spinal cord using 182 \nthe same surgical procedures and post-operative care described above. 183 \n 184 \nElectromyography and nerve stimulation 185 \nTo record the electrical activity of muscles (EMG, electromyography), we directed pairs of Teflon-186 \ninsulated multistrain fine wires (AS633; Cooner Wire Co., Chatsworth, CA, USA) subcutaneously from two 187 \nhead-mounted 34-pin connectors (Omnetics Connector Corp., Minneapolis, MN, USA). Two wires, stripped 188 \nof 1–2 mm of insulation, were sewn into the belly of selected forelimb/hindlimb muscles for bipolar 189 \nrecordings. The head-mounted connectors were fixed to the skull using dental acrylic and four to six screws. 190 \nWe verified electrode placement during surgery by stimulating each muscle through the appropriate head 191 \nconnector channel to assess the biomechanically desired muscle contraction. During experiments, EMG 192 \nsignals were pre-amplified (×10, custom-made system), bandpass filtered (30–1,000 Hz) and amplified (100–193 \n5,000×) using a 16-channel amplifier (model 3500; AM Systems, Sequim, WA, USA). EMG data were 194 \ndigitized (5,000 Hz) with a National Instruments (Austin, TX, USA) card (NI 6032E), acquired with custom-195 \nmade acquisition software and stored on computer. Five forelimb muscles were implanted bilaterally: biceps 196 \nbrachii (BB, elbow and shoulder flexor), extensor carpi ulnaris (ECU, wrist dorsiflexor), flexor carpi ulnaris 197 \n(FCU, wrist plantarflexor), latissimus dorsi (LD, shoulder retractor), and the long head of the triceps brachii 198 \n(TRI, elbow and shoulder extensor). Ten hindlimb muscles were implanted bilaterally: biceps femoris anterior 199 \n(BFA, hip extensor), biceps femoris posterior (BFP, hip extensor and knee flexor), iliopsoas (IP, hip flexor), 200 \nlateral gastrocnemius (LG, ankle plantarflexor and knee flexor), medial gastrocnemius (MG, ankle 201 \nplantarflexor and knee flexor), sartorius anterior (SRT, hip flexor and knee extensor), semitendinosus (ST, 202 \nknee flexor and hip extensor), soleus (SOL, ankle plantarflexor), tibialis anterior (TA, ankle dorsiflexor), and 203 \nvastus lateralis (VL, knee extensor). 204 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nFor bipolar nerve stimulation, pairs of Teflon-insulated multistrain fine wires (AS633; Cooner Wire Co., 205 \nChatsworth, CA, USA) were passed through a silicon tubing. A horizontal slit was made in the tubing and 206 \nwires within the tubing were stripped of their insulation. The ends protruding through the cuff were knotted to 207 \nhold the wires in place and glued. The ends of the wires away from the cuff were inserted into four-pin 208 \nconnectors (Hirose or Samtec) and fixed to the skull using dental acrylic. Cuff electrodes were directed 209 \nsubcutaneously from head-mounted connectors to the left and right SR nerves at the wrist which are purely 210 \ncutaneous at these levels. 211 \n 212 \nExperimental design 213 \nWe collected EMG and kinematic data before and at different time points after staggered hemisections 214 \nduring quadrupedal locomotion at the cat’s preferred treadmill speed (0.3-0.5 m/s). Cats KA and KI stepped 215 \nat 0.3 and 0.5 m/s, respectively, while the other five cats stepped at 0.4 m/s. The treadmill consisted of two 216 \nindependently controlled belts 130 cm long and 30 cm wide (Bertec) with a Plexiglas separator (130 cm long, 217 \n7 cm high, and 1.3 cm wide) placed between the two belts to prevent limbs impeding each other. In the 218 \nintact, preoperative state, cats were trained for 2-3 weeks in a progressive manner, first for a few steps and 219 \nthen for several consecutive minutes, using food and affection as rewards. Once cats could perform 3 -4 220 \nconsecutive minutes, we started the experiments. During experiments, we delivered trains of electrical stimuli 221 \nconsisting of three 0.2 ms pulses at 300 Hz using a Grass (West Warwick, RI, USA) S88 stimulator. At the 222 \nstart of the experiment, we determined the motor threshold, defined as the minimal intensity that elicited a 223 \nsmall motor response in an ipsilateral flexor muscle (e.g., ST or TA) during the swing phase. We then set 224 \nstimulation intensity at 1.2 times the motor threshold to mainly activate large diameter Aβ cutaneous 225 \nafferents. A locomotor trial lasted 4-5 min and consisted of ∼60 stimuli delivered pseudo-randomly every 2–4 226 \ncycles. Stimuli were delivered at specific points of the stimulated forelimb (left or right) movement: mid-227 \nstance, the transition from stance-to-swing, mid-swing and the transition from swing-to-stance. At the start of 228 \nthe experiment, stimulation delays were determined using real-time EMG to detect the onset of an extensor 229 \nburst in relation to stance and swing phases. We then set delays in relation to this extensor EMG so that 230 \nstimuli were delivered at the four desired time points. The timing of the stimuli was assessed during  off-line 231 \nanalysis and stimuli that did not fall in the desired phases were excluded. We characterized responses in 232 \nmuscles of the stimulated forelimb (homonymous), the opposite forelimb (crossed), the hindlimb on the same 233 \nside (homolateral) and the diagonal hindlimb (diagonal). Figure 1A describes the timeline of data collection 234 \nin all seven cats at two time points after the first hemisection (H1T1 and H1T2 in 7 cats) and/or at 1-2 time 235 \npoints after the second hemisection (H2T1 in 2 cats and H2T2 in 6 cats). Some cats only have one time 236 \npoint after the second hemisection because they took longer to recover quadrupedal locomotion. No data 237 \nwere collected for cat KI after the second hemisection due to technical issues with the impl ants.  238 \n 239 \nHistology 240 \nAfter confirming euthanasia (i.e., no cardiac and respiratory functions), we harvested an approximately 2 241 \ncm long section of the spinal cord centered on the lesions. Segments of the dissected spinal cord were then 242 \nplaced in a 25 ml 4% paraformaldehyde (PFA) solution (in 0.1 M phosphate‐buffered saline (PBS), 4°C). 243 \nAfter 5 days, we placed the spinal cord in a new PBS solution containing 30% sucrose for 72 h at 4°C, then 244 \nfroze it in isopentane at -50°C for cryoprotection. The spinal cord was then sliced in 50 µm coronal sections 245 \nusing a cryostat (Leica CM1860, Leica BioSystems Inc., Concord, ON, Canada) and mounted on gelatinized-246 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\ncoated slides. The slides were dried overnight and then stained with a 1% cresyl violet acetate solution for 247 \n12 min. We washed the slides for 3 min in distilled water before being dehydrated in successive baths of 248 \nethanol (50%, 70% and 100%, 5 min each) and transferring them in xylene for 5 min. Dibutylphthalate 249 \npolystyrene xylene was next used to mount and dry the spinal cord slides before being scanned by a  250 \nNanozoomer 2.0-RS (Hamamatsu Corp., Bridgewater, NJ, USA). We then performed qualitative and 251 \nquantitative analyses to estimate lesion extent using ImageJ by selecting the slide with the greatest 252 \nidentifiable damaged area. Using the scarring tissue stained with cresyl violet acetate, we estimated lesion 253 \nextent by dividing the lesion area by the total area of the selected slice and expressed it as percentage. 254 \nLesion extent estimations for individual cats after the first and second spinal lesions are shown in Figure 1B. 255 \nThey ranged from 40.7% to 66.4% (49.2 ± 8.9%) and 33.5% to 53.7% (46.0 ± 7.6%) for the first and second 256 \nhemisections, respectively. 257 \n 258 \nReflex analysis 259 \nWe described the reflex analysis in several of our publications (Hurteau et al., 2017, 2018; Hurteau & 260 \nFrigon, 2018; Merlet et al., 2020, 2021; Mari et al., 2023), and recently illustrated it in detail in (Mari et al., 261 \n2024). Briefly, for all locomotor sessions, EMG signals were low-pass filtered (250 Hz) to facilitate the 262 \nvisualization of the EMG activity envelope. We first defined locomotor cycles from successive burst onsets of 263 \nan extensor from the stimulated forelimb, then separated them as stimulated (i.e., cycles with stimulation) or 264 \ncontrol (i.e., cycles without stimulation) cycles. Sections where the cat stepped irregularly were removed 265 \nfrom analysis based on EMG and video data. Stimulated cycles were then sorted and divided into 4 266 \nsubphases based on stance onset of the stimulated forelimb: swing-to-stance, mid-stance, stance-to-swing 267 \nand mid-swing. Control (C̅) cycles were averaged and rectified to provide a baseline locomotor EMG, an 268 \nindication of the excitability level of the motor pool at stimulation. We averaged the stimulated (S̅) cycles and 269 \ntime normalized C̅ to S̅ cycle durations and superimposed them. To determine response onsets and offsets, 270 \ndefined as prominent positive or negative deflections away from C̅, we set windows using previous studies 271 \nas guidelines (Duysens & Stein, 1978; Duysens & Loeb, 1980; Pratt et al., 1991; Loeb, 1993; Hurteau et al., 272 \n2017, 2018; Hurteau & Frigon, 2018; Mari et al., 2023) with 97.5% confidence intervals. We termed short-273 \nlatency (7–18 ms; SLR) excitatory and inhibitory responses as P1 and N1 responses, respectively, based on 274 \nthe terminology introduced by (Duysens & Loeb, 1980). Responses in the crossed, homolateral, and 275 \ndiagonal limbs that had an onset ≤18 ms were classified as P1 or N1, as the minimal latency for spino -bulbo-276 \nspinal reflexes in the cat is 16-18 ms (Shimamura & Livingston, 1962; Shimamura et al., 1990). Mid-latency 277 \n(19–34 ms; MLR) excitatory and inhibitory responses were termed P2 and N2, respectively. Long-latency 278 \n(35–60 ms; LLR) excitatory and inhibitory responses were termed P3 and N3, respectively. The EMG of 279 \nreflex responses S̅ was then integrated and subtracted from the integrated C̅ in the same time window to 280 \nprovide a net reflex value. This net reflex value was then divided by the integrated C̅ value to evaluate reflex 281 \nresponses. This division helps identify if changes in reflex responses across the cycle are independent of 282 \nchanges in C̅ activity (Matthews, 1986; Frigon & Rossignol, 2007, 2008, 2009; Hurteau et al., 2017, 2018; 283 \nHurteau & Frigon, 2018; Mari et al., 2023, 2024).  284 \n 285 \nStatistical analysis 286 \nWe performed statistical tests with IBM SPSS Statistics V26 (IBM Corp., Armonk, NY, USA).  We 287 \nquantified reflex responses in five forelimb muscles (BB, ECU, FCU, LD, and TRI) and in ten hindlimb 288 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nmuscles (BFA, BFP, IP, LG, MG, SRT, SOL, ST, TA, and VL) when stimulation was delivered to the left or 289 \nright SR. To evaluate whether homonymous, crossed, homolateral and diagonal responses were modulated 290 \nby phase, we performed a one factor (phase) ANOVA on all responses (P1, P2, P3, N1, N2 and N3) in eac h 291 \ncat and state/time point. Because we have several responses within a given phase, we considered all 292 \nresponses during a locomotor session as a population. In our statistical analysis, we used mixed models to 293 \ndeal with incomplete data sets. For instance, reflex responses are sometimes absent after spinal lesions. 294 \nResponse occurrence probabilities, defined as the fraction of evoked responses obtained out of all cats for 295 \npooled SLR, MLR and LLR from the different states/time points were compared using a generalized linear 296 \nmixed model (GLMM) with a binomial distribution and a logit link (mixed logistic regression) in all four limbs. 297 \nThe GLMM analysis was performed using state/time point as a fixed factor. We incorporated random 298 \nintercepts at two distinct levels to consider the hierarchical relationships present in our dataset. A random 299 \nintercept on individual cats at the upper level captured variability across cats. A random intercept on muscle 300 \nnested within cat at a lower level, acknowledging that the same muscle response data were repeatedly 301 \nmeasured within each cat to help us account for any correlation or non-independence of observations within 302 \nthe same cat-muscle pair. Statistical significance for all tests was set at p < 0.05.  303 \n 304 \nRESULTS 305 \nRecovery of quadrupedal locomotion and changes in fore-hind coordination after staggered 306 \nhemisections  307 \nWe recently described changes in the quadrupedal locomotor pattern after staggered thoracic lateral 308 \nhemisections (right T5-T6 followed by left T10-T11), including six cats of the present study (Audet et al., 309 \n2023). Briefly, we showed that cats spontaneously recovered quadrupedal locomotion following both 310 \nhemisections but required balance assistance after the second one. After the first and second hemisections, 311 \nthe coordination between the forelimbs and hindlimbs became weaker and displayed 2:1 patterns, where the 312 \nforelimbs performed two cycles within one hindlimb cycle. 313 \nAfter the first hemisection (H1), all seven cats regained quadrupedal locomotion on the treadmill within 314 \none to two weeks. We were able to conduct reflex sessions for several consecutive minutes at the first 315 \n(H1T1) and second (H1T2) time points. After the second hemisection (H2), the six cats tested recovered 316 \nquadrupedal locomotion within two to five weeks. However, they required mediolateral balance assistance 317 \nduring reflex sessions, which was provided by an experimenter holding the tail of the animal but without 318 \nproviding weight support. As stated in the Methods, some cats only have one time point after the second 319 \nhemisection due to the longer recovery of quadrupedal locomotion and their ability to maintain it for reflex 320 \ntesting. Only two cats, KA and JA, participated in reflex sessions at the first time point (H2T1), i.e., 321 \napproximately two weeks after the second hemisection. All six cats performed reflex sess ions eight weeks 322 \nlater at the second time point (H2T2).  323 \n 324 \nCutaneous reflexes evoked by stimulating the superficial radial nerve before and after staggered 325 \nhemisections 326 \nTo assess the reorganization of cutaneous reflex pathways from forelimb afferents after SCI, we 327 \nstimulated the left and right SR nerves before and after the first and second hemisections and recorded 328 \nreflex responses during quadrupedal treadmill locomotion in muscles of the four limbs. Figures 2-5 illustrate 329 \nexamples from representative cats for homonymous, crossed, homolateral and diagonal responses in 330 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nselected muscles at four phases of the cycle (swing-to-stance transition, mid-stance, stance-to-swing 331 \ntransition and mid-swing). We observed several changes in reflex responses after the first and second 332 \nhemisections, but for the responses shown in Figures 2-5, we only highlight the most noticeable 333 \nobservations. Tables 1-3 provide the full details of reflex responses before and after staggered hemisections 334 \nfor individual cats. For each state/time point, filled areas represent evoked responses and are optimized for 335 \ndisplay according to the strongest response obtained in one of the four phases. If an area is not filled, it 336 \nmeans that the stimulated EMG did not deviate sufficiently from the baseline EMG to be defined as a 337 \nresponse. The scale is optimized per state/time point and differs across state/time points. This is to show the 338 \npattern of evoked responses and its phase-dependent modulation at a given state/time point, as in our 339 \nrecent study (Mari et al., 2024) 340 \n 341 \nHomonymous responses in forelimb muscles. We stimulated the left and right SR nerves and recorded 342 \nhomonymous responses in muscles of the left and right forelimbs, respectively, before and after staggered 343 \nhemisections (Fig. 2 and Table 1). We illustrate homonymous reflex responses in three muscles (ECU, TRI 344 \nand BB) bilaterally in representative cats. The ECU and TRI muscles are mostly ac tive during stance while 345 \nBB is active during swing and/or at the stance-to-swing transition. We observed that the burst profiles of the 346 \nthree selected forelimb muscles remained similar across states (before and after hemisections) and time 347 \npoints during the locomotor cycle.  348 \nIn the left ECU (Fig. 2A, left panel), in the intact state, we observed homonymous P1/P2 responses at 349 \nmid-swing, and weak N1 responses in the other three phases followed by small P3 responses at mid-stance 350 \nand stance-to-swing. After the first hemisection, at H1T1 and H1T2, P1/P2 responses remained at mid-351 \nswing, but relatively strong P2 and/or P3 responses appeared at swing-to-stance and mid-stance. After the 352 \nsecond hemisection, at H2T1, P2/P3 responses were reduced at mid-swing and absent in the other phases. 353 \nAt H2T2, P1/P2 responses recovered at mid-swing and returned at swing-to-stance and mid-stance. In the 354 \nright ECU (Fig. 2A, right panel), in the intact state, we observed P1 responses in all phases followed by N2 355 \nat swing-to-stance and mid-stance and P2 at mid-swing. At H1T1 and H1T2, P1 responses were prominent 356 \nin all phases followed by prominent P2 and/or P3 responses, except for stance-to-swing with only P1 at 357 \nH1T2. At H2T1, P1/P2 responses remained at swing-to-stance and mid-swing, with N1 at mid-stance and no 358 \nresponses at stance-to-swing. At H2T2, we observed P1 responses at mid-swing and stance-to-swing, and 359 \nN1 responses at mid-stance, with no P2/P3 responses in all phases. 360 \nIn the left TRI (Fig. 2B, left panel), we observed homonymous P1 and P2 responses at swing-to-stance 361 \nand P2 responses at mid-swing and mid-stance. At H1T1, P1/P2 and P2 responses remained at swing-to-362 \nstance and mid-stance, respectively, but P2 responses were lost at mid-swing. At H1T2, we N1 responses 363 \nfollowed by P3 responses appeared at swing-to-stance and mid-stance. At H2T2, we only observed P2 364 \nresponses in all phases. In the right TRI (Fig. 2B, right panel), we observed P2/P3 responses in all phases 365 \nthat followed N1 responses at swing-to-stance and mid-stance. At H1T1, N1 responses were lost or 366 \nweakened at swing-to-stance and mid-stance, respectively, but P2 responses remained. N2 responses 367 \nappeared at stance-to-swing and P1 responses at mid-swing, while P2/P3 remained in both phases. At 368 \nH2T1, a weak N1/P2 responses returned or remained stance-to-swing and mid-stance but excitatory 369 \nresponses were lost or reduced at stance-to-swing and mid-swing. At H2T2, N1 responses were prominent 370 \nat swing-to-stance and mid-stance, but P2 responses were lost or reduced. At stance-to-swing and mid-371 \nswing prominent P2/P3 returned.  372 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nIn the left BB (Fig. 2C, left panel), the most noticeable response was a prolonged N1 response at mid -373 \nswing in the intact state. At H1T1, this N1 response was lost and a prominent P3 response appeared. At 374 \nH1T2, the N1 response returned followed by a P3 response. These N1/P3 responses remained at H2T1 and 375 \nH2T2. The other noticeable change was at swing-to-stance, with the appearance of P1 responses at H1T2, 376 \nwhich remained at H2T1 and H2T2. At H2T1 and H2T2, P2/P3 responses also appeared. In the right BB 377 \n(Fig. 2C, right panel), in the intact state, the most noticeable response at mid-swing was an N2 response 378 \nthat followed a brief P1 response. At H1T1, this N2 response was lost while the P1 response became 379 \nprominent. The N2 response returned at H1T2 following a brief P1. At H2T1 and H2T2, the N2 res ponse was 380 \nprominent and was followed by prominent P3 responses. In the other phases, the only noticeable changes 381 \nwere the appearance of a prominent P1 responses at H1T1 at swing-to-stance, which disappeared at H1T2 382 \nbefore returning at H2T2. 383 \nTo summarize, even though thoracic hemisections did not directly affect the pathways transmitting 384 \ncutaneous afferent inputs from the SR nerve to ipsilateral (homonymous) motor circuits in the cervical cord, 385 \nwe observed several small reflex changes in forelimb muscles. 386 \n 387 \nCrossed responses in forelimb muscles. We stimulated the left and right SR nerves and recorded crossed 388 \nresponses in muscles of the right and left forelimbs, respectively, before and after staggered hemisections in 389 \nthe four phases (Fig. 3 and Table 2). We defined the four phases according to the stimulated limb, but it is 390 \nimportant to consider the phase of the contralateral limb where the responses were recorded. 391 \nIn the left ECU (Fig. 3A, left panel), we observed crossed P2 responses in all four phases in the intact 392 \nstate, with the largest at mid-stance of the stimulated right forelimb. After the first and second hemisections, 393 \nP2 responses remained in all phases. In the right ECU (Fig. 3A, right panel), in the intact state, we observed 394 \ncrossed P2 responses in all phases, with the largest at stance-to-swing and mid-swing of the stimulated left 395 \nforelimb. After the first hemisection, P2 responses were reduced at stance-to-swing and mid-swing and P1 396 \nresponses appeared at swing-to-stance. P2 responses recovered at H1T2 and were observed in all phases. 397 \nP2 responses persisted after the second hemisection at H2T2, except at mid-stance where they were 398 \nabsent. 399 \nIn the left TRI (Fig. 3B, left panel), in the intact state, we observed crossed N1 responses during mid-400 \nswing followed by P3 responses, whereas at mid-stance and stance-to-swing, we observed P2/P3 401 \nresponses. After the first hemisection, at H1T1 and H1T2, the response pattern was largely maintained, but 402 \nP2/P3 responses appeared at swing-to-stance at H1T1 and N2/P3 responses at H1T2. After the second 403 \nhemisection, at H2T2, we observed N2 responses at swing-to-stance and mid-swing, maintained P2/P3 404 \nresponses at stance-to-swing and a weak P2/P3 response at mid-stance. In the right TRI (Fig. 3B, right 405 \npanel), in the intact state, we observed prolonged crossed N1 responses at mid-swing and swing-to-stance 406 \nand a P2 response at mid-stance. After the first and second hemisections, we observed prolonged N1 407 \nresponses at swing-to-stance followed by P3 responses, but only at H1T2 and H2T1. At mid-stance, we 408 \nobserved P2/P3 responses at H1T1 and then only P3 responses at H1T2, H2T1 and H2T2. At stance-to-409 \nswing, we observed P2 responses at H1T1, P2/P3 at H1T2, P3 at H2T1 and P2/P3 at H2T2.  At mid-swing, 410 \nN1 or N2 responses were followed by P3 responses at all time points. 411 \nIn the left BB (Fig. 3C, left panel), in the intact state, we observed crossed P2/P3 responses in all four 412 \nphases that peaked at mid-stance of the stimulated right forelimb, which was followed by N3 responses at 413 \nmid-stance only. After the first and second hemisection, P2/P3 responses were maintained in all phases 414 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nexcept at stance-to-swing where they were present at H1T1 and H1T2 but disappeared after the second 415 \nhemisection. In the right BB (Fig. 3C, right panel), in the intact state, we observed crossed P2/P3 responses 416 \nin all phases that peaked at mid-swing of the stimulated left forelimb. After the first and second hemisection, 417 \nP2/P3 responses were maintained in all phases with the strongest responses observed at mid-swing and 418 \nswing-to-stance. 419 \nTo summarize, although we noted some changes in crossed reflex responses in forelimb muscles after 420 \nstaggered hemisections, they were mostly similar to those observed in the intact state. 421 \nTable 1 summarizes homonymous and crossed reflex response patterns observed in all 5 forelimb 422 \nmuscles bilaterally in 6 and 5 cats for the left and right SR nerve stimulations, respectively, before and after 423 \nstaggered hemisections. Overall, the reflex response patterns that we observed in forelimb muscles 424 \nremained generally similar after hemisections, as did their phase-dependent modulation.  425 \n 426 \nTable 1. Homonymous and crossed reflex responses before and after staggered hemisections. 427 \n Left SR nerve stimulation \n Homonymous responses in left forelimb Crossed responses in right forelimb \nCat State BB ECU FCU LD TRI BB ECU FCU LD TRI \nAR Intact \nH1T1 \nH1T2 \nP1* P2* \nP1* \nP1* P2 \nP1* N2/P2* \nP1* P2 \nP1* P2* \nN1/P1* P2* P3* \nN1/P1* P2* P3* \nN1/P1* P2* \nP2* \nP2* \nP2* \nP1* P2* \nP1 P2* \nP1* P2* \nN1 \nx \nx \nP2 P3* \nP2* \nP2* \nP1* \nP2* \nP2* \nN1* \nN1* \nN1 P3* \nN1* P3* \nN1* \nN1* \nHO Intact \nH1T1 \nH1T2 \nH2T2 \nP2* \nP1* P2* \nP1* P2 \nP1* P2 \nN1/P1* \nN1/P1* \nP1* N2* \nN1/P1* \nN1* P2* P3* \nP1* P3* \nN1/P1* P3 \nN1/P1* P3* \nP1* \nP1* \nP1* \nP1* \nP2 \nP1* P2* \nP1* \nP1* P2 \nP2 \nP2* \nP2 \nP2* \nP2* \nP2* \nP2* \nP2* P3* \nP2* \nP2 \nP2 P3* \nP2* \nx \nN2 P3 \nP3 \nP3* \nP2 \nN2* \nN2* \nN2/P2* P3* \nJA Intact \nH1T1 \nH1T2 \nH2T1 \nH2T2 \nP1* N2* \nP1* P3* \nP1* N2 P3* \nP1* N2/P2* P3* \nN1/P1* P2* P3* \nN1/P1* P2* \nN1/P1* \nN1/P1* \nP1* \nN1/P1* \nN1* P3* \nN1/P1* \nN1/P1* P3* \nN1/P1* P3* \nN1/P1* P2* P3* \nP1* P2* \nP1* \nP1* P2* \nP1* \nP1* \nP1* P2* \nP1* P2* P3* \nP1* P2* P3* \nP1* P2* P3* \nP1* P2* \nP2* \nP2* \nP2 \nP2 \nP2* \nx \nP2* P3* \nP2* P3 \nP2 P3* \nP2* P3 \nP2* \nN1* P2* \nP2* P3* \nP2* \nP2* \nN1 P3* \nP2* \nP3* \nN1/P1* P2* \nP2* P3 \nN1* P2* \nN1* P2* P3* \nN1* P2* P3* \nN1* P3 \nN1* P2 P3 \nKA Intact \nH1T1 \nH1T2 \nH2T1 \nH2T2 \nP1* N2* \nP1* P2* \nP1 P2* \nP1 P2* \nP1* P2* \nN1/P1* P2* P3 \nN1/P1* P2* \nN1/P1* P2* P3* \nN1/P1* \nN1/P1* P2* P3 \nN1/P1* P2* \nN1/P1* P2* \nN1/P1* P2* P3* \nN1* \nN1/P1* P2* \nP1* \nN1/P1* P2* \nN1/P1* P2 \n- \n- \nP1* P2* \nP2* \nN1* P2* P3* \nN1 P3 \nP1* P2* P3* \nP2* \n- \nP2* \nP2* \nP2* \nP2* \nx \nP2* P3* \nP2* \nP2* \nP2* \nx \nP2 \nP2* \nP2* \nP2 \nx \nP2* \n- \n- \nx \nx \nP2* P3* \nx \nx \nKI Intact \nH1T1 \nH1T2 \nN1/P1* \nP1* P2* \nP1* P2* \nN1/P1* \nN1/P1* P2* P3* \nN1/P1* P2* P3* \nN1/P1* P2* P3* \nN1/P1* P3* \nN1/P1* P3* \nP1* P2* \nP1* P2* \nN1/P1* P2* \nN1/P1* P2* P3* \nN1/P1* P2* \nN1/P1* P2* P3* \nP2* \nP2 \nP2* \nP2* N3* \nP2* N3* \nP2* N3 \nN2/P2* N3* \nP2* N3* \nP2* N3* \nP2 \nP1* P2* \nP2* \nP2* \nP2* N3* \nP2 N3* \nTO Intact \nH1T1 \nH1T2 \nH2T2 \nP2* \nP2* \nP1* \nP1* \nN1 P2* \nP1* P2* P3* \nN1* P2* \nP1* N2* \nN1/P1* P2* P3* \nx \nN1/P1* P2* P3* \nN1* P3* \nP1* P2* \nP1* \nP1* P2* \nP1* \nP1* P2* \nN1/P1* P2 \nN1* P2 P3 \nP2* \nP2* \nx \n- \nx \nP2* \nP1* P2 \nP2 \nP2* \nP2 N3* \nP2* N3 \nP2* N3* \nP2* N3* \nN1* P2* \nN1* P2 P3* \nP2* P3* \nP2 P3* \nP2* N3* \nP2* \nP2 N3* \nx \n  \nRight SR nerve stimulation  \n Homonymous responses in right forelimb Crossed responses in left forelimb \nCat State BB ECU FCU LD TRI BB ECU FCU LD TRI \nAR Intact \nH1T1 \nH1T2 \nH2T2 \nP1* P2* \nN1/P1* P2* \nP1* P2* P3* \nP1* P2* \nN1/P1* P2* P3* \nN1/P1* P2* P3* \nN1/P1* P2* \nN1/P1* P3* \nN1/P1* P2* P3* \nN1/P1* P2* \nN1/P1* P2* \nN1* P2* \nN1* P2* \nN1* P2* P3 \nN1 P2* P3 \nN1* P2* \nN1/P1* P2* P3* \nN1/P1* P2* P3* \nN1/P1* P2* P3* \nN1/P1* P2* \nN1* P2* \nP2* \nP2* \nP2* P3* \nN1* P2* \nP2* \nP2* P3* \nP2* \nP2* \nP2* \nP2* \nP2* \nN1* \nN1* \nN1* \nN1* \nN1* P2* P3* \nN1* P2* P3* \nN1* P2* P3* \nN1* P2* P3* \nGR Intact \nH1T1 \nH1T2 \nH2T2 \nN1/P1* N2* \nN1/P1* \nN1/P1* \nN1/P1* \nN1/P1* P2* \nN1/P1* P3* \nN1/P1* P3 \nN1* P3* \nN1/P1* P2* \nN1/P1* P3* \nN1/P1* P3* \nN1* P3* \nP1 \nP1* P2* \nP1 \nP1* P2 \nN1/P1* P2* \nN1/P1* P2* P3* \nN1/P1* P3* \nN1/P1* P3* \nP2* \nP2 \nP2 \nP2* \nP2* \nP2* \nP2 \nP2 \nP2* \nP2* \nP2 \nN2/P2* \nP2* \nP2* \n- \n- \nN2* \nP2* \nP2 \nN2/P2* \nJA Intact \nH1T1 \nH1T2 \nH2T1 \nH2T2 \nP1* N2* \nP1* P3* \nP1* N2* P3* \nP1 N2/P2* P3* \nP1* N2/P2* P3* \nP1* \nN1/P1* P2* P3* \nN1/P1* \nN1/P1* \nN1/P1* P2* P3* \nP1* P2* \nN1/P1* P2* \nN1/P1* \nN1/P1* \nN1/P1* P3* \nP1* P2* \nP1* P2* \nP1* \nP1* P2* \nP1* P2* \nP1* N2* P3* \nN1/P1* P2* \nN1/P1* P2* \nP1* P2* \nN1/P1* P2* P3* \nP2* N3* \nP2* N3* \nP2* N3 \nP2* N3* \nP2* N3 \nP2 \nP2* \nP2* \nP2* \nP2* \nP2* \nx \nP2* \nP2* \nP2* \nN1* P3* \nN1 P3* \nP2* \nN1 P3* \nN1 P3* \nP2 \nx \nN2* \nP2* \nP2* P3* \nKA Intact \nH1T1 \nH1T2 \nH2T1 \nH2T2 \nx \nx \nP1* \nP1* P2* \nP1* P2* \nP1* N2* \nP1* P2* \nP1* P2* \nN1/P1* P2* \nN1/P1* P2* \nN1/P1* \nN1/P1* P3* \nN1/P1* P3 \nN1/P1* P2* \nN1/P1* P2* \nP1* P2* \nP1* \nP1* \nx \nx \nP1* N2* \nP1* P2* \nP1* P2* \nP1* P2* \nP1* P2* \nx \nP2* \nx \nP2* \nP2* \nP2* \nx \nx \nP2* \nP2* \nx \nx \nx \nP2* \nP2* \nx \nN1/P1* P3* \nx \n- \n- \nx \nx \nx \nx \nx \nTO Intact \nH1T1 \nH1T2 \nH2T2 \nP1* N2/P2* \nP1* \n- \nP1* \nN1/P1* P2 \nN1* P2 \nN1/P1* P2* \nN1* P2* \nN1/P1* P2 P3* \nx \nP1* \nx \nN1/P1* P2* \nP1* \nP1* P2* \nP1* P2* \nN1* P2 \nN1 P2 P3* \nN1* P2* \nN1* P2 P3* \nx \nP2* \nP2* \nP1* P2* P3* \nP2* \nP2* \nP2* \nN1* P3* \nP2* N3* \nx \nP2* \nP2* P3* \nN1* P3 \nx \nx \nN1* P3* \nN1 P3 \nx \nx \nN1 P3 \n 428 \nHomolateral responses in hindlimb muscles. Out of 10 hindlimb muscles, we show examples from three 429 \nselected muscles, SOL, VL and SRT, bilaterally in representative cats. The SOL and VL muscles are mostly 430 \nactive during stance while SRT is active during swing and/or at the stance-to-swing transition. We define the 431 \nphases relative to the stimulated forelimb. However, after the first and/or second hemisections, cats 432 \nfrequently performed two forelimb cycles within one hindlimb cycle (i.e. 2:1 fore-hind patterns). This means, 433 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nfor example, that stimulation at mid-stance of one forelimb can elicit reflex response at different phases for 434 \nthe hindlimb (see Discussion). 435 \nIn the left SOL (Fig. 4A, left panel), in the intact state, we observed homolateral P2 responses at swing-436 \nto-stance and mid-swing of the stimulated forelimb that were followed by N3 responses at swing-to-stance 437 \nand mid-stance. After the first hemisection, we observed no responses at H1T1, but some recovery at H1T2, 438 \nwith P2/N3 responses at swing-to-stance and mid-stance and P2 responses at mid-swing. Homolateral 439 \nresponses were lost after the second hemisection. In the right SOL (Fig. 4A, right panel), homolateral 440 \nresponse patterns were similar in the intact state, with P2/N3 at swing-to-stance and mid-swing and N3 at 441 \nmid-stance. After the first and second hemisections, we observed no homolateral responses.   442 \nIn the left VL (Fig. 4B, left panel), in the intact state, we observed homolateral P2 responses at mid-swing 443 \nof the stimulated forelimb and N3 responses at mid-stance. After the first and second hemisections, these 444 \nresponses disappeared, with the exception of a weak N3 response at H1T2 swing-to-stance. In the right VL 445 \n(Fig. 4B, right panel), we observed homolateral P2 responses at swing-to-stance and mid-swing of the 446 \nstimulated forelimb. After the first and second hemisections, we observed no responses. 447 \nIn the left SRT (Fig. 4C, left panel), in the intact state, we observed homolateral P2/P3 responses at 448 \nswing-to-stance, mid-stance and mid-swing of the stimulated left forelimb. After the first hemisection, P2/P3 449 \nresponses remained at swing-to-stance and mid-stance at H1T1 but were visibly reduced at H1T2. After the 450 \nsecond hemisection, we observed no responses. In the right SRT (Fig. 4C, right panel), in the intact state, 451 \nwe observed homolateral P2/P3 responses at swing-to-stance, mid-stance and stance-to-swing of the 452 \nstimulated right forelimb. After the first hemisection, P2/P3 responses remained in these phases at H1T1 but 453 \nwere visibly reduced or disappeared at H1T2. After the second hemisection, we observed no responses at 454 \nH2T1 but P2/P3 responses returned at mid-stance and stance-to-swing at H2T2. 455 \n Table 2 summarizes homolateral reflex response patterns in all 10 hindlimb muscles bilaterally in 6 and 5 456 \ncats for the left and right SR nerve stimulations, respectively, before and after staggered hemisections. 457 \nResponse patterns mostly consisted of P2 responses followed by N3 responses in extensors (BFA, LG, MG 458 \nand SOL), and P2/P3 responses in flexors (IP, SRT, ST and TA). Although the phase-dependent modulation 459 \nof responses generally remained after staggered hemisections, when responses were present, we observed 460 \na loss in response occurrence in most muscles after the first and/or second hemisections. However , some 461 \nhomolateral responses returned at H1T2 on the left side. 462 \n 463 \nTable 2. Homolateral reflex responses before and after staggered hemisections. 464 \n Homolateral responses in left hindlimb (left SR nerve stimulation) \nCat State BFA BFP IP LG MG SOL SRT ST TA VL \nAR Intact \nH1T1 \nH1T2 \nN2/P2* N3* \nx \nP2* \nN2/P2* \nx \n- \nP2* \nx \nx \nP2* N3* \nx \nx \nP2* N3* \nx \nP2* \nP2* N2* \nx \nx \nP2* \nx \nx \n- \n- \n- \n- \nx \nx \nP2* \nx \nx \nHO Intact \nH1T1 \nH1T2 \nH2T2 \nN3* \nx \nP1* \nP2* \nx \nx \nx \n- \nP2 \nx \nx \nx \nN3* \nx \nN3* \nx \nP2 N3* \nx \nN3* \nx \nP2 N3* \nx \nN3* \nx \nx \nx \nx \nx \nx \n- \nx \nx \n- \nx \nx \nP2* \nP2 N3 \nx \nN3* \nx \nJA Intact \nH1T1 \nH1T2 \nH2T1 \nH2T2 \nP2* \n- \n- \nx \n- \nP1* \nx \n- \n- \n- \nP2* P3* \nx \nP2* \nN3* \nx \nP2* N3* \nP2 \nP2 N3 \nx \nx \nP2* \nx \nP2* \nx \nx \nP2* N3* \nx \nP2* N3 \nx \nx \nP2* \nP2* \nP2* \nx \nx \nx \nx \n- \n- \n- \nx \nx \nx \nx \nx \nP2* \nx \n- \nx \n- \nKA Intact \nH1T1 \nH1T2 \nH2T1 \nH2T2 \nx \nx \nP2 N3* \n- \n- \nx \n- \nx \n- \n- \n- \n- \n- \n- \n- \nx \nx \nP2 N3* \nx \nx \nP2* N3 \nP2* N3* \nP2* N3* \n- \n- \nP2* N3 \nP2* N3* \nP2* N3 \n- \n- \nx \nx \nx \nx \nx \nx \nx \nP2* \nx \nx \nx \nx \nx \nx \nx \nx \nx \nP2* \n- \n- \nKI Intact \nH1T1 \nH1T2 \nx \nP2* N3* \nP2* \nx \nP2* N3* \nP2* N3* \n- \n- \n- \nN2* \nP2* N3* \nP2* N3* \nN2* \nP2* N3* \nP2* N3* \nN2* \nP2* N3* \nP2* N3* \nx \nP2* \nx \nx \nP1* \nP1* \nx \nx \nx \nx \nP2* N3* \nP2* N3* \nTO Intact \nH1T1 \nH1T2 \nH2T2 \nP2 N3* \nx \nP2* \nx \nP3* \nx \n- \n- \nN2/P2* \nx \nP2* \nx \nP2* N3* \nx \nN3* \nx \nP2* N3* \nx \nP2 N3 \nx \nP2* N3* \nx \nP2* N3* \nx \nP2* \nx \nx \nx \nx \nx \nx \n- \nx \nx \nx \nx \nN3* \nx \nx \nx \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\n  \nHomolateral responses in right hindlimb (right SR nerve stimulation) \nCat State BFA BFP IP LG MG SOL SRT ST TA VL \nAR Intact \nH1T1 \nH1T2 \nH2T2 \nP2* N3 \nx \nx \nx \nx \nx \nx \nx \nP2* \nx \nP2* \nx \nP2 N3* \nN2* \nx \nx \nP2* N3* \nx \nx \nx \nP2* N3* \nN2* \nN2* \nx \nP2* P3* \nP2* \nx \nx \nx \nx \nx \nx \nP2* P3* \nx \nx \n- \nP2* \n- \nx \nx \nGR Intact \nH1T1 \nH1T2 \nH2T2 \nN2* \n- \nx \n- \nx \nx \nP2* \nx \n- \n- \n- \n- \n- \nx \nx \nx \n- \n- \n- \n- \nN2/P2* \nx \nP2 \nx \nP2* \nx \nP2* \nx \n- \n- \nx \nx \nP2* \nP2 \nP2 \nP2* \nN2/P2* \nx \nP2* \n- \nJA Intact \nH1T1 \nH1T2 \nH2T1 \nH2T2 \nx \nx \nx \nx \n- \nx \nx \nx \nx \nx \nx \n- \n- \n- \n- \nx \nx \nx \nx \nx \nx \nx \nx \nx \nx \nP2* N3* \nN2* \nN2 P3* \nx \nx \nP2* \nP2* \nP2* \nx \nP2* \nx \nx \nx \nx \nx \nP2* \nx \nx \nx \n- \n- \nx \nx \nx \nx \nKA Intact \nH1T1 \nH1T2 \nH2T1 \nH2T2 \nx \nx \nx \n- \n- \nx \nx \nx \n- \n- \n- \n- \n- \n- \n- \nx \nx \nx \nx \nx \nx \nP1* \nx \n- \n- \nx \nx \nx \n- \n- \nx \nN1/P1* P2* P3* \nN1/P1* P2* P3* \nP2* \nP1* P2* \nx \nx \nx \n- \nP2* \nx \nx \nx \nx \nx \n- \nx \nx \n- \n- \nTO Intact \nH1T1 \nH1T2 \nH2T2 \nP2* N3* \nx \nx \nx \nx \nx \nx \nx \nP2* N3* \nx \nx \nx \nP2* \nx \nx \nx \nP2* N3* \nx \nx \nx \nP2* N3* \nx \nx \nx \nN1* P3 \nx \nx \nx \nx \nx \nx \nx \nP2* \nx \nx \nx \nP2* \nx \nx \nx \n 465 \nDiagonal responses in hindlimb muscles. In the left SOL (Fig. 5A, left panel), in the intact state, we observed 466 \ndiagonal N2 responses followed by P3 responses at stance-to-swing and mid-swing of the stimulated right 467 \nforelimb, with P2/N3 responses at mid-stance. After the first and second hemisections, we observed no 468 \ndiagonal responses. In the right SOL (Fig. 5A, right panel), in the intact state, we observed diagonal N2 469 \nresponses at mid-stance, stance-to-swing and mid-swing of the stimulated left forelimb followed by P3 470 \nresponses at mid-stance and stance-to-swing. After the first hemisection, at H1T1 and H1T2, N2 responses 471 \nremained at in these three phases but P3 responses disappeared. After the second hemisection, we 472 \nobserved no diagonal responses.  473 \nIn the left VL (Fig. 5B, left panel), in the intact state, we observed diagonal P2 responses at swing-to-474 \nstance and mid-stance of the stimulated right forelimb, with N2 responses at mid-swing. After the first and 475 \nsecond hemisections, we observed no diagonal responses. In the right VL (Fig. 5B, right panel), in the intact 476 \nstate, we observed diagonal P2 responses only at mid-stance of the stimulated left forelimb. After the first 477 \nand second hemisections, we observed no diagonal responses.  478 \nIn the left SRT (Fig. 5C, left panel), in the intact state, we observed diagonal P2 responses in all phases 479 \nexcept at stance-to-swing of the stimulated right forelimb. After the first and second hemisections, we 480 \nobserved no diagonal responses. In the right SRT (Fig. 5C, right panel), in the intact state, we observed 481 \ndiagonal P2 responses in all phases except at swing-to-stance of the stimulated left forelimb. After the first 482 \nhemisection, we observed no responses at H1T1 but P2/P3 reappeared at H1T2 at swing-to-stance and mid-483 \nstance. After the second hemisection, we observed no diagonal responses. 484 \nTable 3 summarizes diagonal reflex response patterns in all 10 hindlimb muscles bilaterally in 6 and 5 485 \ncats for the left and right SR nerve stimulations, respectively, before and after staggered hemisections. 486 \nResponse patterns consisted mostly of N2 followed by P3 responses in extensors (LG and SOL). In flexors 487 \n(BFP, IP, SRT, ST and TA), we observed P2/P3 responses. Similar to homolateral responses, we observed 488 \na loss in response occurrence in most muscles after the first and/or second hemisections , although the 489 \nphase-dependent modulation remained if responses were present. We observed some return of diagonal 490 \nresponses at the second time point after the first hemisection (H1T2) in right hindlimb muscles. 491 \n 492 \nTable 3. Diagonal reflex responses before and after staggered hemisections. 493 \n Diagonal responses in right hindlimb (left SR nerve stimulation) \nCat State BFA BFP IP LG MG SOL SRT ST TA VL \nAR Intact \nH1T1 \nH1T2 \nN2 \nx \nx \nP2* P3* \nx \nx \nP2* \nx \nx \nN2* \nP1* \nx \nN2 \nx \nx \nN2* \nx \nx \nP2* \nx \nx \nP2* \nx \nx \nP2* \nx \nx \nN2* \n- \nx \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nHO Intact \nH1T1 \nH1T2 \nH2T2 \nP2 \nx \nP1* \nP2* \nN1* P2* \nx \nx \nx \nP2* \nx \nP2* \nx \nN2* \nx \nx \nx \nP2* \nx \nP2* N3* \nx \nN2* \nx \nx \nN3* \nx \nx \nx \nx \nx \nx \nx \nx \nP2 \nx \nx \nx \nN2* \nx \nx \nx \nJA Intact \nH1T1 \nH1T2 \nH2T1 \nH2T2 \nx \nx \nP2 \nx \n- \nP3* \nx \nP2 \nx \nx \nP2* \n- \n- \n- \n- \nN2 P3* \nx \nP2 \nx \nx \nP3* \nx \nP2 \nx \nx \nN2* P3* \nN2 \nN2* \nx \nx \nP2* \nx \nP2* \nx \nx \nP3* \nx \nP2* \nx \nx \nx \nx \nx \nx \n- \n- \nx \nx \nx \nx \nKA Intact \nH1T1 \nH1T2 \nH2T1 \nH2T2 \nx \nx \nx \n- \n- \nx \n- \nx \n- \n- \n- \n- \n- \n- \n- \nP2* N3* \nx \nN2/P2* \nx \nx \nx \nx \nx \n- \n- \nN2* \nx \nx \n- \n- \nP2* \nx \nP2* \nx \nx \nx \nx \nP2* \n- \nx \nN2/P2* \nx \nx \nx \nx \n- \nx \nx \n- \n- \nKI Intact \nH1T1 \nH1T2 \nN2* P3 \nP1* P2* \nx \nx \nP1* P2* \nx \n- \n- \n- \nN2 P3 \nN2* \nx \nN2* P3 \nx \nP2* \nN2* P3* \nN2* \nx \nP2 \nP2* \nP2* \n- \n- \n- \nx \nx \nx \nN2 \nx \nN2* \nTO Intact \nH1T1 \nH1T2 \nH2T2 \nN2* P3* \nx \nx \nx \nx \nx \nx \nx \nP2* \nx \nP2* \nx \nP2* N3* \nx \nx \nx \nP2* N3* \nx \nP2 \nx \nx \nx \nN2* \nx \nP2* \nx \nx \nx \nx \nx \nx \nx \nP2 \nx \nx \nx \nP2* \nx \nx \nx \n  \nDiagonal responses in left hindlimb (right SR nerve stimulation) \nCat State BFA BFP IP LG MG SOL SRT ST TA VL \nAR Intact \nH1T1 \nH1T2 \nH2T2 \nx \nx \nx \nx \nP2* \nx \n- \n- \nP2* \nx \nx \nx \nN2* P3* \nN2* \nN2 \nx \nx \nN2* \nN2* \nx \nN2* P3* \nN2* \nN2* \nx \nP2* \nP2* \nP3* \nx \nx \n- \n- \n- \nP2* \nx \nx \n- \nP2* \nN2* \nN2* \nx \nGR Intact \nH1T1 \nH1T2 \nH2T2 \nN2/P2* \nx \nN2* \nx \nx \nx \nx \nx \n- \n- \n- \n- \nN2* \nx \nx \nx \n- \n- \n- \n- \nN2* \nx \nN2* \nx \nP2* \nx \nx \nx \nx \nx \nx \nx \n- \nx \nx \nx \nP2* \nx \nx \n- \nJA Intact \nH1T1 \nH1T2 \nH2T1 \nH2T2 \n- \n- \n- \nx \n- \n- \nx \nx \n- \n- \nP2* \nx \nx \nx \nx \nP3* \nx \nx \nx \nx \nP3* \nx \nx \nx \nx \nN2* P3* \nN2* \nN2* \nx \nN2* \nP2* \nx \nP3 \nx \nx \nx \nx \nx \n- \n- \nP3* \nx \nx \nx \nx \nx \nx \n- \nx \n- \nKA Intact \nH1T1 \nH1T2 \nH2T1 \nH2T2 \nx \nx \nx \n- \n- \nx \nx \nx \n- \n- \n- \n- \n- \n- \n- \nx \nx \nx \nx \nx \nx \nx \nx \n- \n- \nx \nx \nx \n- \n- \nx \nx \nx \nx \nx \nx \nx \nx \nx \nx \nx \nx \nx \nP2* \nP2* \nx \nx \nx \n- \n- \nTO Intact \nH1T1 \nH1T2 \nH2T2 \nP2 \nx \nx \nx \nP2* \n- \n- \n- \nP2* \nx \nx \nx \nN2/P2* P3 \nx \nx \nx \nP2* \nx \nx \nx \nN2/P2* N3/P3* \nx \nx \nx \nP2* \nx \nx \nx \nx \nx \nx \n- \nP2* \nx \nx \nx \nN2/P2* \nx \nx \nx \n 494 \nStaggered hemisections reduce the occurrence of mid- and long-latency responses in hindlimb 495 \nmuscles 496 \nAfter complete or incomplete spinal lesions in cats, mid- and long-latency responses in hindlimb muscles 497 \nare generally reduced or abolished (Fuwa et al., 1991; LaBella et al., 1992; Frigon & Rossignol, 2008; Frigon 498 \net al., 2009; Hurteau et al., 2017; Mari et al., 2024). Here, we investigated the probability of evoking reflex 499 \nresponses in all four limbs before and after staggered hemisections by evaluating the distribution of SLRs 500 \n(N1/P1), MLRs (N2/P2) and LLRs (N3/P3). We did this by calculating the fraction of the total number of SLRs 501 \nor MLRs/LLRs separately, on recorded muscles for each limb across cats. For homolateral and diagonal 502 \nresponses in the left and right hindlimbs, we only evaluated MLRs/LLRs because of infrequent occurrence of 503 \nSLRs. We excluded the H2T1 time point as only two cats were recorded. 504 \nWe found no significant difference in response occurrence probability for left and right homonymous 505 \nSLRs (left, p = .441, GLMM; right, p = .925, GLMM) and MLRs/LLRs (left, p = .496, GLMM; right, p = .401, 506 \nGLMM) across states/time points (Fig. 6A). Similarly, the probability of evoking crossed SLRs (left, p = .085, 507 \nGLMM; right, p = .304, GLMM) and MLRs/LLRs (left, p = .095, GLMM; right, p = .225, GLMM) in left and right 508 \nforelimb muscles did not differ across states/time points (Fig. 6B). In contrast, we found a significant main 509 \neffect of state/time point on homolateral MLRs/LLRs occurrence probability in the left (p = 1.00 × 10-6, 510 \nGLMM) and right (p = 1.50 × 10-5, GLMM) hindlimbs (Fig. 6C). In the left hindlimb, homolateral responses 511 \nwere 8.3 (p = 7.00 × 10-6) and 22.7 (p = 1.25 × 10-4) times more likely to be evoked in the intact state 512 \ncompared to H1T1 and H2T2, respectively. They were also 5.2 (p = 3.73 × 10-4) and 14.4 (p = .001) times 513 \nmore likely to be evoked at H1T2 compared to H1T1 and H2T2, respectively. In the right hindlimb, 514 \nhomolateral responses were 10.0 (p = 1.00 × 10-4), 6.6 (p = 4.16 × 10-4) and 14.7 (p = 6.50 × 10-5) times 515 \nmore likely to be evoked in the intact state compared to H1T1, H1T2 and H2T2, respectively. We found a 516 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nsignificant main effect of state/time point on diagonal MLRs/LLRs occurrence probability in the left (p = 7.12 517 \n× 10-7, GLMM) and right (p = 1.06 × 10-9, GLMM) hindlimbs (Fig. 6D). In the left hindlimb, diagonal 518 \nresponses were 18.2 (p = 8.00 × 10-6), 9.3 (p = 1.46 × 10-4) and 58.8 (p = 7.00 × 10-6) times more likely to be 519 \nevoked in the intact state compared to H1T1, H1T2 and H2T2, respectively. They were also 6.3 (p = .036) 520 \ntimes more likely to be evoked at H1T2 compared to H2T2. In the right hindlimb, diagonal responses were 521 \n28.6 (p = 4.84 × 10-9), 7.2 (p = 1.00 × 10-5) and 100.0 (p = 3.00 × 10-5) times more likely to be evoked in the 522 \nintact state compared to H1T1, H1T2 and H2T2, respectively. They were also 4.0 (p = .009) and 13.8 (p = 523 \n.015) times more likely to be evoked at H1T2 compared to H1T1 and H2T2, respectively. 524 \nTherefore, overall, after the first and second hemisections, the probability of evoking homolateral and 525 \ndiagonal MLRs/LLRs in hindlimb muscles with both left and right SR nerve stimulations was always lower 526 \ncompared to the intact state. In addition, with stimulation of the left SR nerve, the occurrence probability of 527 \nevoking homolateral and diagonal MLRs/LLRS recovered after the first hemisection (at H1T2 compared to 528 \nH1T1), before seeing a drastic decrease after the second hemisection.  529 \n 530 \nDISCUSSION 531 \nIn the present study, we showed changes in reflex responses evoked by electrically stimulating 532 \ncutaneous afferents of the forepaw dorsum (SR nerve stimulation) during locomotion after staggered 533 \nhemisections, extending our recent study with reflex responses evoked by stimulating hindlimb cutaneous 534 \nafferents in the same animals and lesion paradigm (Mari et al., 2024). The main result of the present study 535 \nwas a noticeable loss/reduction of mid- and long-latency homolateral and diagonal responses in hindlimb 536 \nmuscles, after both the first and second hemisections. However, after the first hemisection, we observed a 537 \npartial recovery of these responses evoked by the left SR (contralesional) from the early to the late time 538 \npoint, which then disappeared after the second hemisection. These changes in homolateral and diagonal 539 \nresponses correlated with altered and weakened fore-hind coordination and impaired balance during 540 \nquadrupedal locomotion, as we recently reported (Audet et al., 2023), and also discussed in relation with 541 \nchanges in reflex responses evoked by hindlimb cutaneous afferents (Mari et al., 2024). In the following 542 \nsections, we discuss changes in reflex responses evoked by forelimb cutaneous afferents, the putative 543 \nmechanisms and pathways involved and the functional significance of our results for locomotor 544 \ncontrol/recovery after SCI.  545 \n 546 \nCutaneous reflexes from forelimb afferents before and after staggered lateral hemisections 547 \nIn our staggered lateral hemisections paradigm, the first hemisection unilaterally disrupted direct 548 \ndescending motor pathways from the brain and cervical cord to the lumbar cord, as well as ascending 549 \npathways that carry somatosensory information from the hindlimbs to the cervical cord and then the brain. 550 \nThe second hemisection on the left side then disrupted these direct pathways  contralateral to the first 551 \nhemisection, generating a bilateral disruption. Lesion extent varied between animals (see Fig. 1B) and likely 552 \ncontributed to inter-individual variability. As expected, short- (N1/P1), mid- (N2/P2) and/or long-latency 553 \n(N3/P3) responses in muscles of the homonymous and crossed forelimb remained after the first and second 554 \nhemisections with both left and right SR nerve stimulations (Fig. 6). These responses also retained their 555 \nphase-dependent modulation (Table 1). It is unlikely that the spinal lesions at T5-T6 and then T10-T11 556 \ndamaged forelimb motoneuronal pools, which are located at C5-T2 spinal segments in cats (Sterling & 557 \nKuypers, 1967; Fritz et al., 1986a, 1986b; Hörner & Kümmel, 1993). The SR nerve originates from the 558 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nbrachial plexus, formed by the ventral branches of the last three cervical nerves and the first thoracic nerve , 559 \nand enters spinal segments C7-T1 (Ansón et al., 2013; Mencalha et al., 2014; Hakkı Nur et al., 2020). Thus, 560 \nthe sensorimotor circuits responsible for homonymous and crossed reflex responses in forelimb muscles are 561 \nlargely preserved after thoracic SCIs. 562 \nAlthough the pattern of reflex responses in homonymous and crossed forelimb muscles remained 563 \ngenerally similar after thoracic hemisections, we did observe several small changes ( Figs. 2 and 3). These 564 \nchanges can involve supralesional circuit reorganization (Krupa et al., 2022) and/or the loss of inhibitory or 565 \nexcitatory ascending pathways from lumbosacral segments (Sterling & Kuypers, 1967; Giovanelli Barilari & 566 \nKuypers, 1969; Molenaar & Kuypers, 1978; English, 1985; Alstermark et al., 1987b; Dutton et al., 2006; 567 \nReed et al., 2006). Long ascending propriospinal neurons make direct contact with motoneurons in the most 568 \ncaudal cervical segments in rats (Reed et al., 2006, 2009; Brockett et al., 2013) and C3-C4 segments in cats 569 \n(English, 1985; Alstermark et al., 1987b, 2007), with the vast majority being excitatory (Miller et al., 1973; 570 \nJuvin et al., 2005; Reed et al., 2006; Brockett et al., 2013). These long projecting neurons play a role during 571 \nlocomotion as their silencing can disrupt left-right coordination of the hindlimbs and forelimbs (Pocratsky et 572 \nal., 2020; Shepard et al., 2021). However, fore-hind coordination appears to mainly depend on short 573 \npropriospinal neurons because blocking their activity at thoracic levels, without disrupting transmission in 574 \nlong propriospinal neurons, leads to independent rhythmic activity at cervical and lumbar levels in the in vitro 575 \nneonatal rat preparation (Ballion et al., 2001; Juvin et al., 2005). Moreover, in both fictive and real locomotion 576 \nstudies in decerebrate cats with a complete thoracic transection performed at T10 -T13, reflex responses 577 \nevoked with SR stimulation were preserved and rhythmically modulated in forelimb motoneurons and  578 \nmuscles (Shimamura et al., 1990; Fuwa et al., 1991; Seki & Yamaguchi, 1997). Thus, the neural circuits 579 \nmodulating forelimb reflexes evoked by SR nerve afferents are mainly located at cervical and upper thoracic 580 \nsegments. This can include cervical spinal locomotor CPGs (Yamaguchi, 1992, 2004; Kinoshita & 581 \nYamaguchi, 2001) that interact with primary afferent inputs (Prochazka et al., 2002; Frigon & Rossignol, 582 \n2006; Frigon et al., 2021; Lalonde & Bui, 2021) and supraspinal signals (Shimamura & Livingston, 1963; 583 \nShimamura et al., 1984; Brink et al., 1985; Alstermark et al., 1987a; Fleshman et al., 1988; Fuwa et al., 584 \n1991; Bretzner & Drew, 2005; Ni et al., 2014; Bazley et al., 2014; Duysens, 2024). However, ascending 585 \npathways from the lumbosacral cord likely participate in part of the reflex response patterns and modulation, 586 \nparticularly in the intact state. 587 \nIn hindlimb muscles, the occurrence of homolateral and diagonal mid- and/or long-latency reflex 588 \nresponses was considerably reduced after the first and second hemisections, with both left and right SR 589 \nnerve stimulations (Figs. 4-6). These responses are thus highly dependent on the integrity of descending 590 \npathways running in the thoracic spinal cord. Thoracic lesions directly disrupt descending pathways from the 591 \nbrain and cervical cord that generate homolateral and diagonal reflex responses (Miller et al., 1977; Haridas 592 \n& Zehr, 2003; Hurteau et al., 2018; Mari et al., 2023). Miller and colleagues (1977) proposed that long 593 \ndescending propriospinal neurons, with axons mainly traveling in the ventrolateral spinal cord (Flynn et al., 594 \n2011), constitute the main pathways responsible for homolateral and diagonal responses, although a 595 \ncontribution from short propriospinal neurons is also likely and cannot be excluded . Some of these pathways 596 \nproject ipsilaterally and/or contralaterally (Côté et al., 2018; Laliberte et al., 2019). Additionally, thoracic 597 \nlesions disrupt brainstem pathways that release monoamines throughout the spinal cord and enhance 598 \nneuronal excitability (Noga et al., 2009, 2011). Studies have shown that serotonin facilitates hindlimb 599 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\ncutaneous reflexes in spinal-transected cats (Barbeau & Rossignol, 1990) and rats (Maj et al., 1976; Nozaki 600 \net al., 1977).  601 \nWe observed a partial return of the occurrence of homolateral and diagonal reflex responses evoked by 602 \nthe left SR nerve after the first hemisection, from the first to the second time point, consistent with ongoing 603 \nneuroplasticity (Fig. 6). Thus, over the course of 6-7 weeks, changes occurred in spinal circuits to facilitate 604 \nreflex transmission from cervical to lumbar levels. Numerous studies have shown that the spinal n eural 605 \ncircuitry below an incomplete SCI undergoes substantial reorganization (Barriere et al., 2008; Frigon et al., 606 \n2009; Barrière et al., 2010; Martinez et al., 2011, 2012; Gossard et al., 2015), including after staggered 607 \nlateral hemisections (Jane et al., 1964; Kato et al., 1984, 1985; Stelzner & Cullen, 1991; Courtine et al., 608 \n2008; Van Den Brand et al., 2012; Cowley et al., 2015; Audet et al., 2023). After the first hemisection, the left 609 \nside of the spinal cord remains relatively intact, and plasticity can occur in descending pathways, which can 610 \nbe beneficial or detrimental (Beauparlant et al., 2013; Fink & Cafferty, 2016; Shepard et al., 2023). This can 611 \ninvolve establishing new connections and/or reactivating latent ones, as well as reinforcing spared 612 \ndescending propriospinal and supraspinal pathways through spontaneous collateral sprouting that target 613 \nboth lesioned and unlesioned fibers (Edgerton et al., 2004; Cai et al., 2006; Maier & Schwab, 2006; Fenrich 614 \n& Rose, 2009; Basaldella et al., 2015; Higgin et al., 2020; Zavvarian et al., 2020). The return of neuronal 615 \nexcitability at lumbar levels could have also facilitated responses evoked by descending pathways. 616 \nThe recovery of homolateral and diagonal responses evoked by stimulating the left SR from the early to 617 \nthe late time point after the first hemisection was lost after the second hemisection, which disrupted 618 \ndescending pathways on the left side. This staggered thoracic lateral hemisections paradigm reveals 619 \nlimitations to the formation of new short connections to enable reflex transmission from cervical to lumbar 620 \nlevels. Previous studies have shown that new propriospinal relays can form spontaneously to reroute the 621 \nsupraspinal influences onto lumbar circuits (Bareyre et al., 2004; Courtine et al., 2008; Cowley et al., 2008, 622 \n2010; Zaporozhets et al., 2011; May et al., 2017). However, these new connections appear limited in 623 \nsupporting cutaneous reflex transmission from cervical to lumbar segments. This is consistent with studies 624 \nusing the in vitro neonatal rat brain stem-spinal cord preparation that required neurochemical excitation of 625 \nthoracic propriospinal neurons to generate hindlimb locomotion with brainstem electrical stimulation after 626 \nstaggered thoracic lateral hemisection (Cowley et al., 2008; Zaporozhets et al., 2011). Figure 7 627 \nschematically presents a scenario explaining changes in reflex responses to the four limbs after staggered 628 \nhemisections with left and right SR nerve stimulation.  629 \n 630 \nFunctional considerations 631 \nElectrically stimulating the SR nerve mimics a mechanical contact of the forepaw dorsum, eliciting a 632 \nfunctional response consistent with a stumbling corrective or preventive reaction during the forelimb swing 633 \nand stance phases, respectively, as shown in intact and decerebrate cats (Miller et al., 1977; Matsukawa et 634 \nal., 1982; Drew & Rossignol, 1985, 1987; Shimamura et al., 1990; Fuwa et al., 1991; Hurteau et al., 2018; 635 \nMari et al., 2023). In the present study and other studies, stimulating the SR nerve evoked reflex responses 636 \nin muscles of the four limbs in intact cats, consistent with a whole body functional response to an external 637 \nperturbation to alter limb trajectory and ensure dynamic balance (Haridas & Zehr, 2003; Hurteau et al., 2018; 638 \nPearcey & Zehr, 2019a; Merlet et al., 2022; Mari et al., 2023). Our results indicate that functional responses 639 \nto SR nerve stimulation would have been appropriate in forelimb muscles to alter the trajectory of the 640 \nipsilateral forelimb and reinforce support in the contralateral forelimb after staggered thoracic hemisections, 641 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nas reflex responses in forelimb muscles and their phase modulation were largely preserved . However, the 642 \nloss/reduction of homolateral and diagonal responses suggest an inability to coordinate the hindlimbs, which 643 \nlikely would have affected the whole-body response to a real perturbation.  644 \nThe loss/reduction of homolateral and diagonal responses also reflects a disruption of neural 645 \ncommunication between the brain/cervical cord and the lumbar cord, which cannot be compensated by new 646 \nconnections, as discussed above. This affects sensorimotor functions that depend on this communication. 647 \nIndeed, after staggered thoracic hemisections, we recently showed that fore-hind coordination was altered 648 \nand weakened (Audet et al. 2023). Moreover, after the second hemisection, balance assistance was 649 \nrequired during treadmill locomotion. Although we can only speculate, the loss of cutaneous reflex 650 \ntransmission from cervical to lumbar levels could have contributed to weakened interlimb coordination and 651 \nimpaired postural control. 652 \nIn humans, cutaneous afferents from the arm contribute to locomotor and/or postural control. For 653 \ninstance, cutaneous stimulation at the wrist at the swing-to-stance transition during walking results in 654 \nincreased ankle dorsiflexion (Haridas & Zehr, 2003). This could slow forward progression by reducing 655 \npropulsion from ankle plantarflexors, thereby minimizing a possible collision with an object with the 656 \noutstretched arm. Leg responses evoked from hand cutaneous nerves were facilitated with vision removed 657 \nduring walking, but were restored when participants were asked to lightly touch a stable reference (Forero & 658 \nMisiaszek, 2015) that reinforced stability (Dickstein & Laufer, 2004; Forero & Misiaszek, 2013). Thus, 659 \nforelimb cutaneous afferents can assist with balance during walking. In cats, more weight is distributed to the 660 \nforelimbs for stability and propulsion after spinal lesions (Rossignol et al., 1999) and maintaining proper 661 \nsensorimotor interactions in cervical sensorimotor circuits is important, as shown in the present study .  662 \nAnother factor to consider after single or staggered thoracic lateral hemisections is the emergence of 663 \nspatiotemporal left-right asymmetries between the hindlimbs (Kato et al., 1985; Martinez et al., 2011, 2013; 664 \nAudet et al., 2023), which can lead to walking instability (Dambreville et al., 2015; Huijben et al., 2018). 665 \nRecent studies using split-belt locomotion have shown that inducing left-right asymmetries reduced hindlimb 666 \nreflex responses in some muscles with SR or SP nerve stimulation (Hurteau et al., 2017; Hurteau & Frigon, 667 \n2018; Mari et al., 2023). Split-belt locomotion induces left-right asymmetries in sensory feedback from the 668 \nlimbs, with increased loading for the limbs on the slow belt (Frigon et al., 2015; Park et al., 2019). The 669 \nhemisections also induce left-right asymmetries as the contralesional hindlimb spends a greater proportion of 670 \nthe cycle supporting bodyweight (Martinez et al., 2011, 2012; Audet et al., 2023). This increased loading 671 \ngenerates asymmetric limb sensory feedback, which could play a role in modulating interlimb reflexes.  672 \n 673 \nMethodological limitations 674 \nA limitation of the present study was that balance assistance was required after the second hemisection 675 \nto conduct reflex testing during locomotion, with an experimenter holding the tail for medio -lateral stability, 676 \nbut without providing weight support, as discussed in our recent study (Mari et al., 2024). Without this 677 \nbalance assistance, cats stumbled every few steps. Balance assistance likely facilitated reflex responses 678 \nand their phase-dependent modulation but without it, we could not have conducted reflex testing and 679 \ncompared it to the other states (i.e. intact and following the first hemisection). Another limitation is that we 680 \npooled reflex responses in hindlimb muscles according to the phase of the stimulated forelimb. As stated, 681 \ncats often performed 2:1 fore-hind patterns after the first and second hemisections, where a forelimb 682 \nperformed two cycles within a hindlimb cycle. Because our stimulation is based on the forelimb cycle, it 683 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nmeans that after spinal lesions, some stimuli occurred in different phases of the hindlimb cycle. We 684 \nacknowledge this as a limitation but separating all responses so that both forelimb and hindlimb phases 685 \ncorresponded would have required many more stimuli and several more minutes to complete a session. 686 \nThese studies are difficult to perform in spinal cord-injured cats. Thus, for a given forelimb phase of 687 \nstimulation, we pooled all responses evoked in a hindlimb muscle, irrespective of the phase of the hindlimb. 688 \nThis could have affected average responses, particularly if inhibitory and excitatory responses occurred at 689 \nthe same latency in different hindlimb phases after hemisections. However, we investigated individual 690 \nresponses and observed that most homolateral and diagonal responses were simply lost after hemisections.  691 \n 692 \nConcluding remarks and clinical perspectives 693 \nIn conclusion, the main finding of this study was the loss/reduction of homolateral and diagonal reflex 694 \nresponses in hindlimb muscles from forelimb cutaneous afferents after staggered thoracic hemisections, 695 \nwhich correlated with weakened coordination between the fore- and hindlimbs and impaired balance. How 696 \ndo our findings in the cat model relate to people with spinal cord injury? Our paradigm offers a substrate to 697 \ntest the efficacy of therapeutic approaches (e.g. spinal cord stimulation, pharmacology) to restore neural 698 \ncommunication between the spinal locomotor networks controlling the arms/forelimbs and legs/hindlimbs. 699 \nExploiting cervicolumbar connections, with rhythmic arm movements and/or primary afferent stimulation, 700 \ncould facilitate locomotor rehabilitation after SCI. For instance, in cats (Harnie et al., 2024) and humans 701 \n(Frigon et al., 2004; Zehr et al., 2004; Hiraoka & Iwata, 2006; Loadman & Zehr, 2007; Javan & Zehr, 2008; 702 \nDragert & Zehr, 2009; Hundza & Zehr, 2009; De Ruiter et al., 2010; Hundza et al., 2012; Massaad et al., 703 \n2014; Pearcey & Zehr, 2019b), rhythmic arm movements contribute and/or modulate reflexes in leg/hindlimb 704 \nmuscles. In rats, quadrupedal locomotor training improved the quality of fore-hind coordination after a 705 \nthoracic spinal cord hemisection, with recovery correlating with an increased number of propriospinal 706 \nneurons just above and below the injury site (Shah et al., 2013). In humans with incomplete SCI, 707 \ncoordinating rhythmic arm movements simultaneously with the legs facilitates corticospinal and/or 708 \ncorticofugal drive (Zhou et al., 2017) to leg muscles and modulates cervicolumbar connectivity (Zhou et al., 709 \n2018). Combined arm and leg movements improved locomotor function. Applying transcutaneous spinal cord 710 \nstimulation at the cervical levels can also modulate the activity of lumbar networks (Barss et al., 2020; Parhizi 711 \net al., 2021). 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Modulation of corticospinal input to the legs 1063 \nby arm and leg cycling in people with incomplete spinal cord injury. J Neurophysiol 118, 2507–2519. 1064 \n 1065 \n 1066 \n 1067 \n 1068 \n 1069 \n 1070 \n 1071 \n 1072 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nTables and Figure legends 1073 \nTable 1. Homonymous and crossed reflex response before and after staggered hemisections. The 1074 \ntable shows homonymous and crossed responses (P1, P2, P3, N1, N2 and N3) evoked in individual cats in 1075 \nleft and right forelimb muscles in the intact state, and after the first (H1) and second (H2) hemisections at 1-2 1076 \ntime points (T1 or T2). Bold responses with an asterisk indicate a significant phase modulation (one factor 1077 \nANOVA, p < 0.05). x, No response. -, Non-implanted or non-analyzable (lost or excessive noise) muscle. BB, 1078 \nbiceps brachii; ECU, extensor carpi ulnaris; FCU, flexor carpi ulnaris; LD, latissimus dorsi; TRI, triceps 1079 \nbrachii. 1080 \n 1081 \nTable 2. Homolateral reflex responses before and after staggered hemisections. The table shows 1082 \nhomolateral responses (P1, P2, P3, N1, N2 and N3) evoked in individual cats in left and right hindlimb 1083 \nmuscles in the intact state, and after the first (H1) and second (H2) hemisections at 1-2 time points (T1 or 1084 \nT2). Bold responses with an asterisk indicate a significant phase modulation (one factor ANOVA, p < 0.05). 1085 \nx, No response. -, Non-implanted or non-analyzable (lost or excessive noise) muscle. BFA, biceps femoris 1086 \nanterior; BFP, biceps femoris posterior; IP, iliopsoas; LG, lateral gastrocnemius; MG, medial gastr ocnemius; 1087 \nSOL, soleus; SRT, anterior sartorius; ST, semitendinosus; TA, tibialis anterior ; VL, vastus lateralis. 1088 \n 1089 \nTable 3. Diagonal reflex responses before and after staggered hemisections. The table shows diagonal 1090 \nresponses (P1, P2, P3, N1, N2 and N3) evoked in individual cats in left and right hindlimb muscles in the 1091 \nintact state, and after the first (H1) and second (H2) hemisections at 1-2 time points (T1 or T2). Bold 1092 \nresponses with an asterisk indicate a significant phase modulation (one factor ANOVA, p < 0.05). x, No 1093 \nresponse. -, Non-implanted or non-analyzable (lost or excessive noise) muscle. BFA, biceps femoris anterior; 1094 \nBFP, biceps femoris posterior; IP, iliopsoas; LG, lateral gastrocnemius; MG, medial gastrocnemius; SOL, 1095 \nsoleus; SRT, anterior sartorius; ST, semitendinosus; TA, tibialis anterior; VL, vastus lateralis. 1096 \n 1097 \nFigure 1. Experimental chronology and estimation of lesions extent. (A) Chronology showing the first 1098 \n(T1) and second (T2) experimental time points after the first (H1) and second (H2) hemisections in all cats . 1099 \n(B) For the extent of the lesions, the black area represents the estimation as a percentage of total cross-1100 \nsectional area (reproduced with permission of (Mari et al., 2024)). Note that we only performed one lesion in 1101 \ncat KI. 1102 \n 1103 \nFigure 2. Phase-dependent modulation of cutaneous reflexes evoked in homonymous forelimb 1104 \nmuscles during locomotion before and following staggered hemisections. Each panel shows, from left 1105 \nto right, stance phases of the stimulated forelimb (empty horizontal bars) with its averaged rectified muscle 1106 \nactivity normalized to cycle duration in the different states/time points, and homonymous reflex responses in 1107 \nrepresentative cats for the left and right (A) extensor carpi ulnaris (ECU, cat KA), (B) triceps brachii (TRI, cat 1108 \nTO), and (C) biceps brachii (BB, cat JA). Reflex responses are shown with a post-stimulation window of 80 1109 \nms in four phases in the intact state, and after the first (H1) and second (H2) hemisections at time points 1 1110 \n(T1) and/or 2 (T2). At each state/time point, evoked responses are scaled according to the largest response 1111 \nobtained in one of the four phases. The scale, however, differs between states/time points. The dotted 1112 \nvertical lines in the reflex responses indicate the N1/P1, N2/P2 and N3/P3 time  windows. 1113 \n 1114 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nFigure 3. Phase-dependent modulation of cutaneous reflexes evoked in crossed forelimb muscles 1115 \nduring locomotion before and following staggered hemisections. Each panel shows, from left to right, 1116 \nstance phases of the stimulated forelimb (empty horizontal bars) and crossed forelimb (filled horizontal bars) 1117 \nwith its averaged rectified muscle activity normalized to cycle duration in the different states/time points, and 1118 \ncrossed reflex responses in representative cats for the left and right (A) extensor carpi ulnaris (LECU, cat 1119 \nGR; RECU, cat TO), (B) triceps brachii (LTRI, cat AR; RTRI, cat JA), and (C) biceps brachii (BB, cat JA). 1120 \nReflex responses are shown with a post-stimulation window of 80 ms in four phases in the intact state, and 1121 \nafter the first (H1) and second (H2) hemisections at time points 1 (T1) and/or 2 (T2). At each state/time point, 1122 \nevoked responses are scaled according to the largest response obtained in one of the four phases. The 1123 \nscale, however, differs between states/time points. The dotted vertical lines in the reflex responses indicate 1124 \nthe N1/P1, N2/P2 and N3/P3 time windows. 1125 \n 1126 \nFigure 4. Phase-dependent modulation of cutaneous reflexes evoked in homolateral hindlimb 1127 \nmuscles during locomotion before and following staggered hemisections. Each panel shows, from left 1128 \nto right, stance phases of the stimulated forelimb (empty horizontal bars) and homolateral hindlimb (filled 1129 \nhorizontal bars) with its averaged rectified muscle activity normalized to cycle duration in the different 1130 \nstates/time points, and homolateral reflex responses in representative cats for the left and right (A) soleus 1131 \n(SOL, cat TO), (B) vastus lateralis (LVL, cat HO; RVL, cat TO), and (C) anterior sartorius (SRT, cat JA). 1132 \nReflex responses are shown with a post-stimulation window of 80 ms in four phases in the intact state, and 1133 \nafter the first (H1) and second (H2) hemisections at time points 1 (T1) and/or 2 (T2). At each state/time point, 1134 \nevoked responses are scaled according to the largest response obtained in one of the four phases. The 1135 \nscale, however, differs between states/time points. The dotted vertical lines in the reflex responses indicate 1136 \nthe N1/P1, N2/P2 and N3/P3 time windows. 1137 \n 1138 \nFigure 5. Phase-dependent modulation of cutaneous reflexes evoked in diagonal hindlimb muscles 1139 \nduring locomotion before and following staggered hemisections. Each panel shows, from left to right, 1140 \nstance phases of the stimulated forelimb (empty horizontal bars) and diagonal hindlimb (filled horizontal 1141 \nbars) with its averaged rectified muscle activity normalized to cycle duration in the different states/time 1142 \npoints, and diagonal reflex responses in representative cats for the left and right (A) soleus (LSOL, cat TO; 1143 \nRSOL, cat JA), (B) vastus lateralis (VL, cat TO), and (C) anterior sartorius (LSRT, cat TO; RSRT, cat JA). 1144 \nReflex responses are shown with a post-stimulation window of 80 ms in four phases in the intact state, and 1145 \nafter the first (H1) and second (H2) hemisections at time points 1 (T1) and/or 2 (T2). At each state/time point, 1146 \nevoked responses are scaled according to the largest response obtained in one of the four phases. The 1147 \nscale, however, differs between states/time points. The dotted vertical lines in the reflex responses indicate 1148 \nthe N1/P1, N2/P2 and N3/P3 time windows. 1149 \n 1150 \nFigure 6. Reflex response occurrence in all four limbs before and after staggered hemisections. 1151 \nResponse occurrence probabilities are shown for short- (SLR) and mid-/long-latency (MLRs/LLRs) 1152 \nresponses with stimulation of the left or right superficial radial nerve before (intact) and after the first (H1) and 1153 \nsecond (H2) hemisections at time points 1 (T1) and/or 2 (T2). Tables 1, 2 and 3 provide details on the 1154 \nnumber of pooled data for SLRs, MLRs and LLRs. Each filled circle represents the mean probability ± 1155 \nconfidence interval (95%) in 5 forelimb or 10 hindlimb muscles pooled across cats for homonymous/crossed 1156 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\n(A and B) and homolateral/diagonal (C and D) responses, respectively. If a significant main effect of 1157 \nstate/timepoint was found (generalized linear mixed model), we compared states/time points. Asterisks 1158 \nindicate significant differences at p < 0.05*, p < 0.01** and p < 0.001***. When one state/time point was 1159 \nsignificantly different from two states/time points, the comparison starts with a longer horizontal line. 1160 \n 1161 \nFigure 7. Schematic illustration of putative pathways and mechanisms contributing to cutaneous 1162 \nreflexes and their modulation before and after staggered hemisections.  1163 \nIn the intact state, afferents from the left (A) and right (B) superficial radial (SR) nerves contact spinal 1164 \ninterneurons that project to motoneurons within the hemisegment (homonymous responses) at cervical 1165 \nlevels and commissural interneurons projecting contralaterally (crossed responses). SR nerve afferents also 1166 \nmake contacts with propriospinal neurons that project to lumbar levels, terminating ipsilaterally (homolateral 1167 \nresponses) or contralaterally via collateral projections at different segments (diagonal responses). The 1168 \npathways responsible for short-latency responses (SLRs) are mainly confined to spinal circuits, including 1169 \nSLRs in hindlimb muscles. The pathways contributing to mid- and long-latency responses (MLRs/LLRs) 1170 \ntransmit sensory information to supraspinal structures via long ascending projection neurons (propriospinal 1171 \nand/or dorsal lemniscal pathways) that project back to spinal circuits controlling the fore- and hindlimbs. After 1172 \nthe first hemisection (on the right side), SLRs and MLRs/LLRs in forelimb muscles remain present although 1173 \ntheir response pattern can change due to the loss of inhibitory or excitatory ascending pathways from 1174 \nlumbosacral circuits. The occurrence of MLRs/LLRs in hindlimb decreases (dashed lines) due to disruptions 1175 \nin ascending and descending pathways to and from supraspinal structures. Spared supraspinal axons are 1176 \npotentially strengthened or sprout to form new connections to transmit descending signals. After the second 1177 \nhemisection (on the left side), direct ascending and descending pathways are both disrupted, and 1178 \nreorganization of short propriospinal neurons is required to relay signals through the lesions, although their 1179 \nability to do so is limited, leading to a considerable loss in MLRs and LLRs in hindlimb muscles. 1180 \n 1181 \nFigure Abstract 1182 \nContacting an obstacle during locomotion activates cutaneous afferents to maintain balance and coordinate 1183 \nall four limbs. Spinal cord injuries disrupt neural communications between spinal networks controlling the 1184 \nfore- and hindlimbs, impairing balance and limb coordination. Cutaneous reflex pathways can be used to 1185 \ndevelop therapeutic approaches for restoring ascending and descending transmission to facilitate locomotor 1186 \nrecovery. 1187 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nForelimb \nreflexes \nHindlimb  \nreflexes \nT5-T6\nT5-T6Intact\nT10-T11\nT5-T6\nT10-T11\nSupraspinal\n structures\nFore-hind coordination\nperformanceSuperficial radial nerve stimulation\nExtensor\nFlexor\nP2\nP1 P2\nN1 P3\nP2\nP2 N3\n8 weeks post-SCI \nFigure abstract\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nFigure 1\nst\n1  lesion\nnd\n2  lesion\n0 2 4 6 8 10 12 16 18\nTime post injuries (weeks)\nIntact\nH1T1\nH1T2\nH2T2\nH2T1\n14 20\nCat ID\nAR\nTO\nKI\nKA\nJA\nHO\nGR\nTOKIKAJAHOGRAR\nst\n1  lesion \n(right T5-T6)\nnd\n2  lesion \n(left T10-T11)\n53.1\n46.9\n42.266.443.547.440.751.4\n33.5 40.9 49.4 51.7 53.7\nA\nB\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nLeft ECU \nRight BBLeft BB\nRight ECU\nLeft forelimb homonymous responses Right forelimb homonymous responses\nSTIM 10 ms\n1 2\n43\nSTIM 10 ms\n2\n4\n1\n3\nSTIM 10 ms\n1 2\n43\nSTIM 10 ms\n2\n4\n1\n3\nA\nB\n2\n3\n4\nstance-to-swing\nmid-swing\nmid-stance\n1 swing-to-stance\nLeft TRI\nSTIM 10 ms\n1 2\n43\nC\nRight TRI\nSTIM 10 ms\n1 2\n43\n1\n11\n1\n1 1\n1\n11\n1\n1 1\n2\n22\n2\n2 2\n4\n44\n4\n4 4\n3\n33\n3\n3 3\nIntact\nH1T1\nH1T2\nH2T1\nH2T2\nFigure 2\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nLeft ECU \nRight BBLeft BB\nRight ECU\nLeft forelimb crossed responses Right forelimb crossed responses\nSTIM 10 ms\n1 2\n43\nSTIM 10 ms\n1 2\n43\nSTIM 10 ms\n2\n4\n1\n3\nSTIM 10 ms\n1 2\n43\nstance-to-swing\nmid-swing\nmid-stance\nswing-to-stance\nLeft TRI\nSTIM 10 ms\n1 2\n43\n11 2 43\n11 2 43 1\n1\n11\n1\n1\n11\n2\n2\n22\n4\n4\n44\n3\n3\n33\nRight TRI\nSTIM 10 ms\n2\n4\n1\n3\nIntact\nH1T1\nH1T2\nH2T1\nH2T2\n2\n3\n4\n1\nFigure 3\nA\nB\nC\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nLeft SOL\nRight VLLeft VL\nRight SRTLeft SRT\nRight SOL\nLeft hindlimb homolateral responses Right hindlimb homolateral responses\nSTIM 10 ms\n1 2\n43\nSTIM 10 ms\n1 2\n43\nSTIM 10 ms\n2\n4\n1\n3\nSTIM 10ms\n1 2\n43\nSTIM 10 ms\n1 2\n43\nSTIM 10 ms\n1 2\n43\n1 2\n21 44 3\n2 34 1\n21 43\n23 4 1\n4 3 1 24 3\nstance-to-swing\nmid-swing\nmid-stance\nswing-to-stance\n1st cycle 2nd cycle\n4\n21 4\n23 4\n1 24 3\n4\n1 43\n12 23\n1 24 3\n4 21 43\n23 4 1\n1 24 3\n4\n3\n4\n2\n13 21 4\n23 4\n1 24 3\n13\nIntact\nH1T1\nH1T2\nH2T1\nH2T2\n2\n3\n4\n1\nFigure 4\nA\nB\nC\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nLeft SOL\nRight VLLeft VL\nRight SRTLeft SRT\nRight SOL\nSTIM 10 ms\n1 2\n43\nSTIM 10 ms\n1 2\n43\nSTIM 10 ms\n1 2\n43\nSTIM 10 ms\n2\n4\n1\n3\nSTIM 10 ms\n1 2\n43\nSTIM 10 ms\n2\n4\n1\n3\nstance-to-swing\nmid-swing\nmid-stance\nswing-to-stance\n12 2\n2\n22 4 1\n4 3\n1st cycle 2nd cycle\n3\n21 44\n22 3\n12 243\n13\n21 43\n23 31\n1 22 43\n4\nLeft hindlimb diagonal responses Right hindlimb diagonal responses\n1 44 3\n12 2\n2\n22 4 1\n4 3\n3\n1 44 3\n12 2\n2\n22 4 1\n4 3\n3\n1 44 3\n4\n4\n21 44\n22 3\n12 243\n134\nIntact\nH1T1\nH1T2\nH2T1\nH2T2\n2\n3\n4\n1\nFigure 5\nA\nB\nC\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nOccurrence probability Occurrence probability\nSLR\nSLR\n0 0.5 1.0\n0 0.5 1.0\n0 0.5 1.0\n0 0.5 1.0\n0 0.5 1.0\n***\n***\n***\n**\n0 0.5 1.0\n0 0.5 1.0\n*********\n0 0.5 1.0\nLeft SR stimulation Right SR stimulation Intact\nH1T1\nH1T2\nH2T2\n*********\n**\n* *\n*********\nHomonymous \nCrossed \nHomolateral Homolateral \nDiagonal Diagonal \nCrossed \nHomonymous A\nB\nC\nD\nMLR/\nLLR\nMLR/\nLLR\nMLR/\nLLR\nMLR/\nLLR\nFigure 6\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint \n\nIntact state After first hemisection\nMLR/LLRSLRMotoneuron\nExcitatory neurons Inhibitory neurons Propriospinal neurons Modulatory effects\nAfter second hemisectionA\nB\nLumbar\nThoracic\nThoracic\nCervical\ncollaterals\nSupraspinal structures Supraspinal structuresSupraspinal structures\nLumbar\nCervical\nSupraspinal reflex\n control\nSupraspinal reflex\n control\nLemniscal \npathways\nLemniscal \npathways\nPropriospinal \npathways\nPropriospinal \npathways\nAscending/Descending \ncircuits reorganization\nSupraspinal structures Supraspinal structuresSupraspinal structures\nLeft SR \nLeft SR Left SR \nRight SR Right SR \ncollaterals\nRight SR \nFigure 7\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 23, 2024. ; https://doi.org/10.1101/2024.04.23.590723doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}