Evidence of spinal cord comparator modules for rapid corrections of movements

Evidence of spinal cord comparator modules for rapid corrections of movements | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Evidence of spinal cord comparator modules for rapid corrections of movements View ORCID Profile Gorkem M. Ozyurt , Sarah Chiasson , Alex M Laliberte , Filipe Nascimento , Emam Khan , William P. Mayer , Gardave S. Bhumbra , Turgay Akay , View ORCID Profile Tuan V. Bui , View ORCID Profile Marco Beato , View ORCID Profile Robert M. Brownstone , View ORCID Profile Rémi Ronzano doi: https://doi.org/10.1101/2025.08.27.672590 Gorkem M. Ozyurt 1 Department of Neuromuscular Diseases, University College London , London, United Kingdom 2 Department of Neuroscience Physiology and Pharmacology (NPP), University College London , Gower Street, WC1E 6BT London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gorkem M. Ozyurt Sarah Chiasson 3 Brain and Mind Research Institute, Department of Biology, University of Ottawa , Ottawa, ON, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alex M Laliberte 3 Brain and Mind Research Institute, Department of Biology, University of Ottawa , Ottawa, ON, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Filipe Nascimento 1 Department of Neuromuscular Diseases, University College London , London, United Kingdom 2 Department of Neuroscience Physiology and Pharmacology (NPP), University College London , Gower Street, WC1E 6BT London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Emam Khan 3 Brain and Mind Research Institute, Department of Biology, University of Ottawa , Ottawa, ON, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site William P. Mayer 4 Atlantic Mobility Action Project, Brain Repair Centre, Department of Medical Neuroscience, Dalhousie University , Halifax, Nova Scotia, Canada 5 Department of Medical Neuroscience, Dalhousie University , Saint John, New Brunswick, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gardave S. Bhumbra 2 Department of Neuroscience Physiology and Pharmacology (NPP), University College London , Gower Street, WC1E 6BT London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Turgay Akay 4 Atlantic Mobility Action Project, Brain Repair Centre, Department of Medical Neuroscience, Dalhousie University , Halifax, Nova Scotia, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tuan V. Bui 3 Brain and Mind Research Institute, Department of Biology, University of Ottawa , Ottawa, ON, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tuan V. Bui For correspondence: tuan.bui{at}uottawa.ca m.beato{at}ucl.ac.uk r.brownstone{at}ucl.ac.uk r.ronzano{at}ucl.ac.uk Marco Beato 2 Department of Neuroscience Physiology and Pharmacology (NPP), University College London , Gower Street, WC1E 6BT London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marco Beato For correspondence: tuan.bui{at}uottawa.ca m.beato{at}ucl.ac.uk r.brownstone{at}ucl.ac.uk r.ronzano{at}ucl.ac.uk Robert M. Brownstone 1 Department of Neuromuscular Diseases, University College London , London, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Robert M. Brownstone For correspondence: tuan.bui{at}uottawa.ca m.beato{at}ucl.ac.uk r.brownstone{at}ucl.ac.uk r.ronzano{at}ucl.ac.uk Rémi Ronzano 1 Department of Neuromuscular Diseases, University College London , London, United Kingdom 2 Department of Neuroscience Physiology and Pharmacology (NPP), University College London , Gower Street, WC1E 6BT London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rémi Ronzano For correspondence: tuan.bui{at}uottawa.ca m.beato{at}ucl.ac.uk r.brownstone{at}ucl.ac.uk r.ronzano{at}ucl.ac.uk Abstract Full Text Info/History Metrics Preview PDF Abstract Successful movement requires continuous adjustments in response to changes in internal and external environments. To do so, neural circuits continuously compare efference copies of motor commands with sensory input to respond to sensory prediction errors. Some responses need to be very fast and, for limbs, likely occur in as yet undefined spinal cord circuits. Here, we describe spinal circuits involving dI3 neurons, showing that they receive multimodal sensory inputs and direct efferent copies from both Renshaw cells and motor neurons. We further show that they form connections to motor pools, including diverging connections to antagonist motor nuclei. Reducing dI3 neuronal activity diminished stumbling responses, as did disrupting Renshaw cell circuits, providing evidence for a comparator role of dI3 neurons for online corrections. Together, our findings reveal a pivotal role for dI3 neurons functioning as comparators of internal predictions and external sensory feedback to mediate rapid corrections of ongoing movements. Introduction We all make mistakes. Fortunately, nervous systems have evolved to take corrective actions in response to unexpected perturbations and avoid them when possible. Furthermore, corrective action to avoid potentially devastating consequences is often needed with minimal delay. For example, without immediate corrective action, an unexpected obstacle during gait could lead to a fall and result in the loss of prey, becoming prey, or as is the case for humans, serious injury or death. In fact, falls are the second leading causes of accidental death in the world, with 79 people dying per hour from falls, and many others (about 4,250 per hour) being significantly injured 1 . It is thus crucial to understand how nervous system circuits are wired in such a way that the incidence of serious errors is minimised. Principles of movement correction have previously been outlined, with control theory providing a solid foundation 2 . These principles can be applied over many time scales – from milliseconds to days. The fundamental mechanisms underlying motor adaptation and learning have been extensively studied in cerebellar systems 3 , 4 . To perform motor tasks, the nervous system generates specific motor commands, and the outcomes of these motor commands are continually evaluated in light of feedback provided by multiple sensory modalities 5 . This multimodal sensory feedback (“instructive inputs”) is integrated with processed efference copies of the motor commands (“predictive inputs,” predicting the sensory consequences of the movement) by “comparator” neurons, which generate corrective signals to optimize the completion of the intended movement 6 – 10 . While the brain is undoubtedly key for adaptation and learning, it may be too remote (in distance and synapses) from the centres that control spinal motor neurons to immediately (in milliseconds) correct fundamental errors during ongoing movement. Spinal cord circuits have the capacity to integrate multimodal sensory feedback to generate immediate motor responses through corrective reflexes. In fact, interneurons receiving multiple sensory modalities were identified long ago 11 . But for these neurons to serve as comparators in order to respond to unpredicted perturbations, they would also need to receive efference copy as inputs. These inputs, particularly during gait – a movement generated by spinal cord circuits – would need to originate within the spinal cord for immediate corrections to occur. That is, in many instances of reflexive corrections, such as stumbling correction, the generated corrections, occurring with delays of milliseconds, are dependent on the motor dynamics 12 – 14 . Therefore, there must be spinal cord circuits that integrate motor commands with sensory inputs to provide corrections based on current motor dynamics. We have previously shown that dI3 neurons – spinal neurons born in the 3 rd dorsal spinal progenitor domain and defined by their expression of the transcription factor Isl1 15 – mediate grasp reflexes and are important for plasticity of spinal circuit function following spinal cord transection 16 , 17 . At least a subset of this cardinal class, in addition to having sensory inputs, receives inputs from locomotor circuits and some of them innervate motor neurons 16 , 18 . Therefore, they are well positioned as candidates to integrate predictive and instructive inputs, and to provide the substrate for dynamic corrections of ongoing movements. But do they function as comparator neurons responsible for error correction? To map dI3 neurons circuitry and assess their function in corrective responses, we used a combination of mouse genetics, viral tracing, physiological methods, and behavioural challenges. We show that individual dI3 neurons receive instructive inputs from multimodal sensory modalities as well as inputs from Renshaw cells and motor neurons. Furthermore, individual dI3 neurons innervate flexor, extensor, or both sets of motor neurons. We then show that dI3 neurons mediate corrective responses. Lastly, we provide converging evidence that the direct input from Renshaw cells to dI3 neurons functions to facilitate immediate corrections of movements. We propose that Renshaw cells thus model the direct efference copy transmitted by motor axons. Taken together, we suggest that a subset of dI3 neurons functions as spinal cord comparator neurons to facilitate fast corrective actions extending the definition of comparator systems from the brain to local spinal circuits. Results dI3 neurons receive multimodal sensory feedback segregated along their somato-dendritic compartment Having previously shown that dI3 neurons receive direct primary afferent input from low threshold cutaneous and proprioceptive sensory neurons 16 , we now sought to further define the distribution of primary afferent inputs to dI3 neurons. In the lumbar cord, dI3 neurons are distributed in the intermediate laminae from the medial to lateral grey matter (( Supplemental figure 1A-C , 16 ), with the most medial population restricted to the caudal lumbar cord (( Supplemental figure 1D ). A large proportion of dI3 neurons are located in a zone, previously defined as a region of convergence 19 , through which proprioceptive afferents from multiple muscles project ( Figure 1A ). We therefore sought to determine whether there was preferential targeting of dI3 neurons from proprioceptive afferents in this region. To do so, we used vGlut1, a marker of low-threshold primary afferents and corticospinal projections, together with parvalbumin (PV) that labels proprioceptive afferents in the postnatal spinal cord 20 . Download figure Open in new tab Supplemental figure 1: dI3 neurons segregate spatially based on sensory inputs they receive but not based on their passive properties. (A) Representative example of a lumbar transverse hemisection at P11 showing dI3 neurons (tdTom ON ChAT OFF , in magenta ON blue OFF ). (ii) Part of the transversal section contained in the dashed box in (i) showing dI3 neurons (tdTom ON ChAT OFF ) at a higher magnification. (B-C) Spatial (B) distribution and (C) density of all dI3 neurons (in magenta) and motor neurons (in blue) on an idealized lumbar transverse section. (D) Spatial density showing the distribution of the same population as in B-C along the rostro-caudal axis with a front view of the cord from L3 to L6. (E) Example of a dI3 neurons (tdTom ON ) with proprioceptive boutons (vGlut1 ON -PV ON -tdTom ON ) apposed to its soma. (F) Percentage of lumbar dI3 neurons with somatic proprioceptive (Pro, vGlut1 ON -PV ON -tdTom ON ) vs cutaneous (Cut, vGlut1 ON -PV OFF -tdTom ON ) boutons apposed to their soma. (G-H) Spatial distribution of dI3 neurons colour coded by the number of (G) proprioceptive or (H) cutaneous boutons apposed to their soma scaled by their cross sectional area. (I) Spatial distribution of dI3 neurons colour coded by the cross section of their soma. (J-K) Medio-lateral distribution of (J) input resistance and (K) cell capacitance of dI3 neurons along the medio-lateral axis. Inset in (K) describes the coordinate system used to measure the medio-lateral position of recorded neurons. (L-M) Comparison of the size of the DR-evoked excitation and inhibition (L) raw and (M) scaled to cell conductance. Box plots are accompanied by the mean difference on the right, n shows the number of plotted root responses from L4 and L5. (N) DR-evoked excitation jitter vs latency colour coded by the age of the animal used, the dashed line indicates the cut-off for monosynaptic window based on Doyle and Andresen (2001). (A-I) The same n=3 animals were used as in figure 1A-E . (A-D) Data shown are from L3 to L6 pooled from the 3 animals, (E-I) n =192 dI3 neurons analysed. (J-K) Recording obtained in (J) n=77 and (K) n=73 dI3 neurons from the same preparation than in figure 1J-O and figure 2E-H .(L-N) Recordings obtained from the same n=5 animals, 2 roots and 37 dI3 neurons than in figure 1J-O . Plotted values in L and M are restricted to root stimulations that triggered both excitatory and inhibitory measurable responses in the recorded dI3 neurons to allow hierarchically bootstrapped paired samples Wilcoxon tests and paired Hedge’s G. Download figure Open in new tab Figure 1: dI3 neurons receive spatially segregated multimodal sensory afferences and inhibitory inputs from sensory relays. (A) Representative example of a lumbar transverse section of a Isl1 cre/wt ; Ai14 fl/wt P11 mouse. The box highlights the region of convergence of proprioceptive afferences where many dI3 neurons are localized. (B) Experimental strategy used to label proprioceptive and cutaneous boutons from the distal hindlimb with CTB injections in the GS and hind paw. (C) Example of two dI3 neurons (tdTom ON ) with proprioceptive boutons labelled from the distal hindlimb (CTB ON -vGlut1 ON -PV ON ) apposed to their soma. (D) Number of proprioceptive boutons from the distal hindlimb (CTB ON -vGlut1 ON -PV ON ) apposed to dI3 neurons soma. (E) Spatial distribution of dI3 neurons colour coded by the number of somatic proprioceptive boutons traced from the distal hindlimb. (F) Experimental strategy used to label the dendritic arborization of lumbar dI3 neurons and mechanosensory neurons terminals from the sural nerve. (G) Representative example of a lumbar transverse section with terminal endings from the sural nerve labelled with CTB (in magenta), dI3 neurons labelled by membrane tagged YFP (in green) and vglut1 (in blue). The region within the dashed box in (i), is shown at higher magnification in (ii).The boxed region in ii is expanded in iii-v, showing a CTB ON -vGlut1 ON bouton apposed to a dorsal dendrite of a dI3 neuron and shown in a 3D reconstruction in vi. (H) Examples of reconstructed dI3 dorsal dendrites with apposed vGlut1 ON (in blue) and CTB ON -vGlut1 ON (in magenta) terminals. (I) Position of dI3 neurons with terminals from the sural nerve (CTB ON -vGlut1 ON ) apposed to their dorsal dendrites. Dots in magenta correspond to the two examples showed in (H). (J) Schematic of the spinal cord preparation that was used to record dorsal root (DR)-evoked excitation and inhibition (K) of dI3 neurons which were held at −75 mV and 0 mV in voltage-clamp configuration to record excitatory and inhibitory responses respectively. (L-M) Distribution of the size of DR-evoked excitation (L) and inhibition (M) based on the location of dI3 neurons along medio-lateral axis. Inset in (L) describes the coordinate system used to measure the medio-lateral position of recorded neurons. (N-O) Comparisons of inhibition and excitation (N) latency and (O) jitter, where n indicates the number of root stimulations (L4 and L5) plotted. (A-E) Tissues from n=3 animals and n =192 dI3 neurons were analysed, (F-I) Tissues from n=2 animals. (J-N) N=5 mice (P6-11), stimulation of L4 and L5, and recording from 37 dI3 neurons, plotted values in (N-O) are restricted to root stimulations that triggered both excitatory and inhibitory responses in the recorded dI3 neurons where (N) latency and (O) jitter were measurable, to allow non-hierarchically bootstrapped paired samples Wilcoxon tests and paired Hedge’s G. We found that more than 80% of dI3 neurons were apposed by a variable number of PV ON /vGlut1 ON somatic proprioceptive synaptic boutons (( Supplemental figure 1E-G ). We further observed the greatest density of somatic proprioceptive boutons in the interposed position along the medio-lateral axis, the region of convergence of proprioceptive axons. Since Isl1 is also expressed in the vast majority of sensory neurons, but not in corticospinal neurons 16 , 21 , we further characterised vGlut1 ON /tdTom ON /PV OFF boutons as terminals from putative cutaneous sensory neurons. We observed that cutaneous boutons were rarely found on the soma of dI3 neurons, with about 7% of dI3 neurons (13/192) having somatic cutaneous boutons. Furthermore, dI3 neurons showing somatic cutaneous afferences were located in the most medial or dorso-lateral part of the dI3 neuron distribution and largely devoid of somatic proprioceptive boutons (( Supplemental figure 1F, H ). To target more specifically sensory neurons from the distal limb, we next traced sensory axons retrogradely using cholera toxin subunit B (CTB). Co-injections were performed into the gastrocnemius muscle and subcutaneously into the hind paw to visualize both proprioceptive and cutaneous fibres ( Figure 1B ). We found that proprioceptive boutons (CTB ON /vGlut1 ON /PV ON ) were again mostly restricted to the soma of dI3 neurons in the interposed area ( Figure 1C-E ). Conversely, cutaneous afferents labelled from the hind paw were rarely found on dI3 neuron somata with only 2/192 dI3 showing CTB ON /vGlut1 ON /PV OFF boutons, consistent with the position of dI3 neurons being ventral to the region where cutaneous afferents have been shown to terminate 22 , 23 . Yet we previously showed that dI3 neurons receive afferent input from low-threshold mechanoreceptors and mediate low-threshold cutaneo-motor disynaptic reflexes 16 . We therefore hypothesized that the different modalities of sensory afferents could segregate onto specific subcellular domains of dI3 neurons with the cutaneous afferences projecting specifically to their dorsally-projecting dendrites. To test this hypothesis, we combined intraspinal injection of AAV1-flex-mYFP to induce the expression of a membrane tagged YFP for visualization of dI3 dendritic arbours with retrograde tracing from the sural nerve to label cutaneous afferents ( Figure 1F ). We first observed that while the vast majority of dI3 somata are restricted to lamina V-VII (( Supplemental figure 1A-C , 16 ), their dendrites extend dorsally, densely invading lamina III-IV (with few dendrites even reaching lamina II; Figure 1G ). While only 1/50 dI3 somata had cutaneous boutons from the sural nerve in apposition, 100/101 boutons were observed in apposition to dI3 dorsal dendrites (n=2 animals, Figure 1G-H ). Therefore, cutaneous inputs are segregated to the dorsal dendrites of dI3 neurons including dendrites of the interposed dI3 neurons that also receive somatic proprioceptive inputs ( Figure 1I ). We then studied functional connectivity of primary afferents to dI3 neurons through patch-clamp recording of dI3 neurons during dorsal root stimulation at low threshold intensities in an isolated spinal cord preparation. Cross-sectional area of dI3 neurons seemed to increase from medial to lateral, but input resistance and whole cell capacitance were not associated with their position (( Supplemental figure 1I-K ). Following DR stimulation, we observed both excitatory (36/37) and inhibitory (32/37) responses in recorded dI3 neurons. The amplitudes of both conductances ( Figure 1J , ( Supplemental figure 1L-M ) were similar to each other and similar along the medio-lateral axis ( Figure 1J-L ). Both the latency and the jitter of excitation were lower than those of the inhibitory responses recorded ( Figure 1M-N ), with some of the excitatory responses (even at this young age and room temperature) clearly in the monosynaptic window in keeping with our anatomical observations (( Supplemental figure 1N , 24). In summary, cutaneous inputs are dendritic, while proprioceptive sensory inputs densely innervate the somata of dI3 neurons interposed along the medio-lateral axis. Furthermore, stimulation of mixed proprioceptive (from flexor and extensor muscles) and low threshold cutaneous afferents leads to both direct excitation and indirect (likely disynaptic) inhibition of these neurons. Together, this organisation suggests that there may be gating of sensory modalities to dI3 neurons by spinal inhibitory neurons during movement. dI3 neurons receive an efferent copy of motor outputs While sensory feedback (instructive input) is essential for motor control, and several spinal populations of dorsal neurons have been shown to rely on mechanosensory afferences to modulate corrective reflexes 25 – 27 , the integration of efferent copies (predictive input) with the sensory feedback improves the ability of sensory-motor circuits to estimate the current state of limbs and dynamically adapt the task while avoiding instabilities 3 , 6 , 28 – 30 . To do so, the predictive input would be a “negative image” of the efferent command 31 , 32 . We previously showed that dI3 neurons receive inhibitory inputs from locomotor circuits and suggested that these inputs could represent an efference copy transmitted to dI3 neurons 17 . We now sought to determine whether dI3 neurons integrate predictive with instructive inputs. Ideal candidates to convey a direct efference copy within spinal circuits would be Renshaw cells (a subpopulation of V1 neurons; 33 ), as these inhibitory neurons receive direct input from motor neuron axons – the most fundamental efferent information 10 . Using En1 cre/wt mice crossed with Ai34d fl/wt mice to induce tdTomato expression at the presynaptic terminals of all V1 interneurons ( Figure 2A ), we could visualize Renshaw cell axon boutons by their expression of calbindin and tdTomato ( Figure 2B , 34 , 35 ). We found that about 50% of dI3 neurons had tdTom ON /Calbindin ON boutons apposed to their somata, and that these boutons were mostly restricted to the interposed region of the dI3 population (region of convergence) as well as those ventral to this region ( Figure 2C-D ). Download figure Open in new tab Figure 2: dI3 neurons receive efferent copies from Renshaw cells and motor neurons. (A) Experimental strategy to visualize presynaptic boutons from Calbindin ON V1 interneurons on dI3 somata. (B) Representative example of a lumbar transverse section showing Isl1 (in grey) at P8. The blue arrowhead highlights the dI3 neuron showed in (ii) at a higher magnification with a Calbindin (in green), tdTomato (in magenta) double positive boutons apposed to its soma (neurotrace staining in blue). The dashed box highlights the presynaptic boutons at higher magnification in (iii-v) with the colocalization of calbindin and tdTomato apposed to the membrane (neurotrace staining) of a dI3 neuron. (C) Percentage of dI3 neurons (Isl1 ON ) with Calbindin ON V1 boutons apposed to their soma. Each data point represents the average of one animal. (D) Spatial distribution of dI3 neurons colour coded by the number of Calbindin ON V1 boutons apposed to their soma. (E) Schematic of the longitudinal preparation of the ventral lumbar cord used to record recurrent excitation and inhibition from dI3 neurons. (F) Sample traces of voltage clamp recording showing recurrent inhibition (red, holding potential 0 mV) and excitation (blue, holding potential −75 mV). (G-H) Comparisons of inhibition and excitation (G) latency and (H) jitter following VR-stimulation where n indicates the number of L4 and L5 responses plotted. (I) Schematic representing the connectivity of Renshaw cells, motor neurons and dI3 neurons, together with the type of recording configuration used to test the effect of mecamylamine on VR-stimulation responses. (J) Sample responses obtained from a Renshaw cell (red, loose cell attached recording), motor neuron (blue, whole-cell recording) and dI3 neuron (black, whole-cell recording) after ventral root stimulation in control condition. (K) Same responses recorded after 100 µM mecamylamine application into the recording solution. (L-M) Comparisons of the recurrent inhibition size in control vs 100 µM mecamylamine conditions recorded from (L) motor neurons and (M) dI3 neurons. (C-D) N=3 animals between P7-P8, n= 273 dI3 neurons analysed. (G-H) N=8 mice (P5-15), L4 and L5 roots stimulated and 40 dI3 neurons, plotted values are restricted to root stimulations that triggered both excitatory and inhibitory responses in the recorded dI3 neurons where (G) latency and (H) jitter were measurable to allow hierarchically (G) and non-hierarchically (H) bootstrapped paired samples Wilcoxon tests and paired Hedge’s G analysis. (J-M) n= 10 Renshaw cells were recorded from N=4 glyT2-eGFP animals, while n=6 dI3 neurons and n=4 motor neurons were recorded in a total of N=3 and 4 Isl1 cre/wt ;Ai14 fl/wt animals respectively. (L, M) Non-hierarchically bootstrapped paired samples Wilcoxon tests and paired Hedge’s G. Since Renshaw cells are not the only V1 interneurons expressing calbindin in the spinal cord 36 , we proceeded to record dI3 neurons identified by tdTomato expression while stimulating ventral roots (VR) to activate motor axons ( Figure 2E ). In our reduced (isolated ventral lumbar spinal cord) preparations, we found that about 75% (n=30/40) of dI3 neurons received VR evoked inhibition of varying magnitude ( Figure 2F , ( Supplemental figure 2A-C ). Intriguingly, the majority of dI3 neurons (88%, n=35/40) also received VR-evoked excitation (purely glutamatergic) suggesting possible direct innervation of dI3 neurons by motor neurons ( Figure 2F , ( Supplemental figure 2A-C, E-G ). This observation was corroborated by both shorter latency and lower jitter observed for excitation than inhibition, with most of the excitatory responses falling into or close to the clear monosynaptic window 24 especially when taking into account the effect of age on these parameters ( Figure 2G-H ; ( Supplemental figure 2D ; 37 ). To further confirm that the VR-evoked inhibition of dI3 neurons was indeed mediated by Renshaw cells, a non-competitive antagonist of nicotinic acetyl-choline receptors, mecamylamine, was used to block cholinergic transmission from motor neurons to Renshaw cells 38 , 39 . Following mecamylamine application, Renshaw cell spiking activity (cell-attached recordings) following VR-stimulation was either abolished (6/10 cells) or the latency and jitter increased significantly (4/10 cells, Figure 2I-K ). We found that mecamylamine led to a 35% decrease of the inhibitory currents recorded from dI3 neurons following VR stimulation, similar to the decrease of recurrent inhibition of motor neurons in response to VR stimulation (∼25%, Figure 2I-K ), further validating the identification of dI3 neurons as a post-synaptic target of Renshaw cells. Download figure Open in new tab Supplemental figure 2: dI3 neurons receive monosynaptic purely glutamatergic inputs from motor neurons. (A) Schematic of the preparation used to record ventral root (VR)-evoked responses. (B-C) Comparison of the size of recurrent inhibition and excitation conductance, (B) raw and (C) scaled to cell conductance where n is the number of root stimulations (L4 and L5) recorded. (D) Plot indicating recurrent excitation jitter vs latency. Dashed line indicating the cut-off for monosynaptic window based on Doyle and Andresen (2001). (E-F) Representative traces for recurrent excitation (E) in control conditions and (F) in the presence of glutamate receptor antagonists. (G) The size of the recurrent excitation in control and glutamate-blocked conditions. (B-D) Recordings obtained from the same 8 mice (P5-15), 2 roots and 40 dI3 neurons than in Figure 2G-H . In B and C, plotted values are restricted to root stimulations that triggered both excitatory and inhibitory measurable responses in the recorded dI3 neurons to allow non-hierarchically bootstrapped paired samples Wilcoxon tests and paired Hedge’s G. (G) n=10 root stimulations in 5 animals (P7-15) and 6 dI3 neurons. Our results show that dI3 neurons receive efferent copies of motor commands, via inhibitory inputs from Renshaw cells as well as through direct collaterals from motor neurons. Taken together, our observations suggest that dI3 neurons are well positioned to combine sensory feedback and efferent copies of motor outputs to mediate fast corrections of motor commands. dI3 neurons innervate motor neurons for flexion, extension, or co-activation We next sought to quantify the projections of dI3 neurons to motor neurons: such projections – to the lateral motor column and to a lesser extent to medial motor neurons – have previously been described 16 , 18 , 40 . However, the identification of the motor neuron pools innervated was restricted to the pool innervating the quadriceps 18 . Therefore, we used Isl1 cre/wt ; Ai34d fl/wt animals, restricting tdTomato expression to the presynaptic terminals of Isl1 ON neurons, combined with retrograde labelling of motor neurons innervating the ankle flexor, tibialis anterior (TA) or the ankle extensor, gastrocnemius (Gs, Figure 3A ). We quantified tdTom ON /vGlut2 ON presynaptic boutons apposed to retrogradely labelled motor neurons; these terminals would not be from vGlut2 ON sensory neurons as the latter neurons project only to the dorsal horn 41 , 42 . We confirmed that motor neurons from the medial column receive only a few boutons from dI3 neurons, while motor neurons innervating both Gs and TA receive numerous dI3 terminals ( Figure 3B,C ; 40 ). Furthermore, the number of dI3 presynaptic terminals apposed to flexor motor neurons (TA) was significantly higher than those apposed to extensor motor neurons (Gs, Figure 3C ). Since the expression of vGlut2 by motor neurons has been reported but remains unclear 43 , 44 , we used Chat cre/wt ; Ai34d fl/wt animals to validate that presynaptic boutons quantified as dI3 terminals were not confounded with recurrent innervation between motor neurons 45 . We observed only a few tdTom ON /vGlut2 ON boutons from cholinergic neurons apposed to lateral motor neurons, which confirmed that the vast majority of terminals identified in the Isl1 cre/wt ; Ai34d fl/wt animals were from dI3 neurons (( Supplemental figure 3A-C ). Download figure Open in new tab Supplemental figure 3: Interposed di3 neurons are responsible for most of motor neurons innervation. (A) Representative example of a motor neuron (ChAT, in blue) with one tdTom (in magenta), vGlut2 (in green) double positive bouton apposed to its soma in a ChAT cre/wt ; Ai34d fl/wt mouse at P11. The double positive bouton apposed to the soma is highlighted with the dashed box in i, shown at higher magnification in ii-iv. Filled arrowhead shows the tdTom ON vGlut2 ON bouton and contour arrowhead the other tdTom ON vGlut2 OFF boutons. (C) Number of tdTom ON -vGlut2 ON double positive presynaptic boutons apposed to the soma of motor neurons from the lateral motor column scaled by their cross sectional area. (D) Representative example of a lumbar transverse hemisection from a Isl1 cre/wt ; Ai14 fl/wt mouse at P11 showing dI3 neurons (tdTom ON ChAT OFF , in magenta) stained with an antibody against Isl1 (in green). (ii-iii) Part of the transversal section contained in the dashed box in (i) showing dI3 premotor neurons (tdTom ON ChAT OFF ) at a higher magnification, with about half of them still expressing Isl1 (in green). (E) Percentage of dI3 neurons (tdTom ON ChAT OFF ) labelled by immunohistostaining on Isl1 (tdTom ON ChAT OFF Isl1 ON ) at P11 in the lumbar cord. (F) Spatial density showing the distribution of all dI3 (in magenta), dI3 neurons immunoreactive for Isl1 (in green) and motor neurons (in blue) from L3 to L6 with a front view of the cord. (G-H) Spatial (G) distribution and (H) density of the same population than in (F) on an idealized cross section of the lumbar spinal cord. (I-J) Spatial (I) distribution and (J) density of ipsilateral premotor dI3 neurons innervating TA (in magenta) and LG (in green) on an idealized hemi-section. (K) Spatial distribution of dI3 neurons premotor to TA and LG along the rostro-caudal axis with a front view of the lumbar spinal cord. (C) n=9 LMC motor neurons in N=3 animals. (D-H) Data shown are from L3 to L6 pooled from N=3 animals and collected on the same samples as in Figure Supplemental 1A-D. (I-K) Same dataset than Figure 3E-L , n=5 animals pooled. Download figure Open in new tab Figure 3: dI3 to motor neuron innervation is wired to mediate any type of motor output. (A) Experimental strategy to visualize presynaptic boutons from dI3 neurons (tdTom ON -vGlut2 ON ) onto motor neurons innervating the tibialis anterior (TA) or the gastrocnemius (GS). CTB intramuscular injections were performed at P8 and the tissues collected at P11. (Bi) Representative example of a motor neuron innervating TA (CTB ON , in grey) with tdTom ON (in magenta), vGlut2 ON (in green) double positive boutons apposed to its soma (ChAT, in blue). The double positive boutons apposed to the soma are highlighted with red asterisk or in the dashed box showed at higher magnification (ii-iv). (C) Number of tdTom ON -vGlut2 ON double positive presynaptic boutons apposed to the soma of motor neurons of the medial column or motor neurons innervating TA or GS scaled by their cross sectional area. (D) Monosynaptic retrograde tracing used to describe dI3 premotor neurons that project to motor neurons innervating the TA (ΔG-RV-mCherry) or the LG (ΔG-RV-GFP). (Ei) Representative example of a lumbar transverse section showing premotor neurons traced from the injection in TA (mCherry, in magenta), LG (GFP in green) and a staining against Isl1 (in grey). (ii-iii) Part of the transversal section contained in the dashed box in (i) showing dI3 premotor neurons (Isl1 ON , in grey) traced from motor neurons innervating the TA (Isl1 ON -mCherry ON ) or the LG (Isl1 ON -GFP ON ). White arrowheads highlight the premotor dI3 neurons. (F) Percent of premotor neurons innervating TA vs LG motor neurons that are identified as dI3 neurons (Isl1 ON ) scaled by the total number of premotor neurons traced from TA and LG injections respectively. (G-H) Spatial (G) distribution and (H) density of motor neurons (in blue), all dI3 neurons (Isl1 ON neurons, in grey) and dI3 neurons premotor to TA and LG motor neurons (respectively in magenta and green). (I) Spatial density showing the distribution of premotor dI3 neurons innervating TA and LG motor neurons along the rostro-caudal axis with a front view of the cord. (J) Orthogonal projection of a lumbar section following a premotor tracing from TA and LG. It shows two dI3 neurons that are projecting to both TA and LG motor neurons (Isl1 ON -mCherry ON -GFP ON , filled arrowheads) and one dI3 neurons innervating LG motor neurons (Isl1 ON -mCherry OFF -GFP ON , contour arrowhead). (K) Percent of premotor dI3 neurons innervating both TA and LG motor neurons per animal. (L) Spatial distribution of dI3 neurons innervating both TA and LG motor neurons. (B-C) n=12 MMC motor neurons in n=6 animals, 18 TA and 22 GS motor neurons in n=5 animals. (E-L) data shown are collected from L3 to L6, and pooled from n=5 animals. Non-hierarchically bootstrapped (C) Kruskal-Wallis test followed by Dunn’s multiple comparisons test and (F) two-tailed Mann Whitney test. We then asked where the premotor dI3 neurons innervating flexor versus extensor motor pools are located, and whether some are wired for co-activation of flexor and extensor muscles. To tackle these questions, we used simultaneous monosynaptic retrograde rabies virus tracing from both extensor (the lateral gastrocnemius, LG) and flexor (TA) muscles. G-deleted rabies viruses were injected into LG and TA muscles of P2 pups expressing the G protein in cholinergic neurons (i.e. in motor but not sensory neurons, preventing anterograde tracing; Figure 3D , 46 , 47 ). Immunoreactivity against Isl1, visible in 40 to 50% of L3-L6 dI3 neurons at this age (( Supplemental figure 3D-E ) largely without spatial bias compared with the whole population of dI3 neurons (( Supplemental figure 3F-H ), was used to identify them ( Figure 3E ). We found that the percentage of lumbar premotor neurons that are flexor-innervating dI3 neurons was greater than those innervating extensor motor neurons (6.7 ± 1.9% from TA vs 3.4 ± 1.3% from LG, Figure 3F ), supporting the flexor bias observed using anterograde tracing ( Figure 3C ). Furthermore, premotor dI3 neurons were segregated to the interposed and most dorso-lateral part of dI3 neurons distribution, with virtually no premotor dI3 neurons in the most medial subpopulation restricted to segments L5 and L6 of the lumbar enlargement ( Figure 3G-H , ( Supplemental figure 3F-H , cf. ( Supplemental figure 1D ). Although there was enrichment of premotor dI3 neurons to TA and LG in the same segments as the innervated motor pool (L4 and L5), premotor dI3 neurons were seen rostral to these segments as well (in L3), showing that some dI3 neurons project descending axons to at least the adjacent segment ( Figure 3I , ( Supplemental figure 3K , 48 ). Lastly, the simultaneous tracing from TA and LG showed that some dI3 neurons innervate both TA and LG motor neurons and are therefore positioned to mediate co-contraction of antagonist muscles ( Figure 3J ). Although the proportion of these divergent premotor dI3 neurons is underestimated ( Figure 3K-L ; 49 ), their proportion (∼8%) was relatively high compared to what we previously described for the whole population of lumbar premotor neurons innervating TA and LG motor pools (∼4%; 49 ). In summary, the position of premotor dI3 neurons spatially overlap with the location of dI3 neurons receiving multimodal sensory feedback and those receiving an efferent copy from Renshaw cells (and motor neurons). Furthermore, they are wired to contribute to flexion, extension, or co-contraction of antagonist muscles. Taken together, dI3 neurons, as a population, show key components of comparator modules. dI3 neurons form cellular comparator modules We next sought to determine whether instructive and predictive inputs converge on the same premotor dI3 neurons. Our first step was to determine whether the muscles innervated by the motor neurons to which dI3 neurons project, supply proprioceptive inputs to the same dI3 neurons. To do so, we used intramuscular monosynaptic rabies tracing (same animals as Figure 3 ), which labels both premotor dI3 neurons and proprioceptive neurons innervating the injected and synergist muscles ( Figure 4A , 46 ) but not afferents from antagonist muscles 50 . Although the proportions are largely underestimated due to the relatively low efficiency of monosynaptic retrograde tracing 49 , we nevertheless found that at least one third of premotor dI3 neurons also receive PV ON /vGlut1 ON /RV ON proprioceptive inputs from homonymous proprioceptive neurons ( Figure 4B-E ). Furthermore, this was the case when tracing from flexor as well as extensor muscles ( Figure 4D, E ). Download figure Open in new tab Figure 4: dI3 neurons show specific properties of cellular comparator modules. (A) Following an injection of ΔG-RV inducing the expression of a fluorescent protein (FP), sensory and motor neurons innervating the injected muscles are infected. The RV can propagate to the premotor neurons (here dI3 neurons), and sensory neurons innervating motor neurons from targeted and synergist muscles, i.e. homonymous and heteronymous motor pools. (B) Example of a representative lumbar transverse section after a first round of staining (sequence 1) used to identify premotor dI3 neurons, showing premotor neurons traced from the injection in TA (mCherry, in magenta), LG (GFP, in green) and a staining against Isl1 (in grey). (C) The same section was then unmounted and reacted a second time (sequence 2) to visualize PV (in blue) and vGlut1 (in grey) and reimaged. After the second staining, the premotor dI3 neuron to TA motor neurons, highlighted in the dashed box in B, shows, a presynaptic bouton from a proprioceptive neuron (vGlut1 ON -PV ON ) traced from the injection in TA (mCherry ON , in magenta). This shows that premotor dI3 neurons are receiving putative inputs from homonymous or heteronymous proprioceptive neurons. (D-E) Number of premotor dI3 to (D) TA and (E) LG motor neurons with apposed boutons from proprioceptive afferences of the homonymous or synergist muscles (vGlut1 ON -PV ON -FP ON bouton). (F) Schematic of the longitudinal preparation of the hemi lumbar cord used to record dorsal root (DR)-evoked as well as ventral root (VR)-evoked excitation and inhibition from dI3 neurons (tdTom ON , in magenta) or non-dI3 excitatory neurons (tdTom OFF GFP OFF , in black) in Isl1 cre/wt ;Ai14 fl/wt and Isl1 cre/wt ;Ai14 fl/wt ;glyT2-eGFP animals. (G-H) Representative traces for (G) DR-evoked and (H) VR-evoked responses recorded from a single dI3 neuron held at Vh −75 mV for excitation and Vh 0 mV for inhibition. (I-J) Numbers of responsive (I) dI3 neurons and (J) non-dI3 excitatory neurons to DR-evoked excitation and VR-evoked inhibition shown as Venn diagrams. (K) Percentage of recorded neurons receiving both DR-evoked excitation and VR-evoked inhibition whether they are identified as dI3 or non-dI3 excitatory neurons (exc non-dI3). (B-E) N=2 animals traced from TA and LG. (G-K) Total of 25 dI3 neurons from 6 animals and 26 non-dI3 excitatory neurons from 3 animals (P5-14). (K) Fisher’s exact test. We next assessed whether individual dI3 neurons receive convergent predictive (Renshaw cell) and instructive (primary afferent) inputs. Following stimulation of dorsal as well as ventral roots on longitudinal hemisected spinal cord preparations ( Figure 4F ), we found that a large proportion of dI3 neurons receive both DR-evoked excitation and VR-evoked inhibition (16/25 recorded dI3 neurons from n=6 animals, Figure 4G-I ). Ten of these also received VR-evoked excitation and DR-evoked inhibition ( Figure 4G-H , ( Supplemental figure 4A-B ). This proportion is likely underestimated since in these preparations, some sensory axons are damaged. Indeed, the percentage of dI3 neurons with dorsal root evoked excitation in hemisected spinal cords is 20% lower (76%, 19/25 cells) than that seen in ventral spinal cord ablated preparations (97%, 36/37 cells), and the amplitude of the excitatory responses is one fifth (292 ± 337% vs 60 ± 57%, Figure 4G ). To determine whether the convergence of instructive and predictive inputs was specific to dI3 neurons or shared by other neuronal populations in the intermediate laminae, we recorded DR and VR-evoked responses in non-dI3 excitatory neurons in preparations from animals expressing tdTomato in dI3 neurons and eGFP in inhibitory neurons ( Figure 4F ). In contrast to dI3 neurons, we observed that only a small proportion of non-dI3 excitatory neurons receive both DR-evoked excitation and VR-evoked inhibition (3/26 recorded excitatory non-dI3 neurons from n=3 animals, Figure 4J-K ), with 2/26 also receiving VR-evoked excitation and DR-evoked inhibition (( Supplemental figure 4C-D ), showing relative specificity of the convergence of instructive and predictive inputs to dI3 neurons. Download figure Open in new tab Supplemental figure 4: dI3 neurons in the intermediate lamina receive a specific set of inputs. (A) Schematic of the lumbar hemicord preparation from Isl1 cre/wt ;Ai14 fl/wt ;glyT2-eGFP or Isl1 cre/wt ;Ai14 fl/wt animals used to record (DR)-evoked as well as (VR)-evoked excitation and inhibition from dI3 neurons (tdTom ON , in magenta) or non-dI3 excitatory neurons (tdTomato OFF GFP OFF , in black). (B-C) Distribution of (B) dI3 neurons and (C) non-dI3 excitatory neurons depending on their responses to VR and DR-evoked stimulations. (D) Percentage of recorded neurons receiving DR and VR-evoked excitation and inhibition whether they are identified as dI3 or non-dI3 excitatory neurons (exc non-dI3). (B-D) Same 25 dI3 neurons and 26 non-dI3 excitatory neurons from respectively n=6 and 3 animals (P5-14) presented in figure 4I-K . (D) Fisher’s exact test. Together, these results suggest that a large proportion of dI3 neurons in the interposed medio-lateral region may form independent cellular comparator modules combining predictive and instructive inputs to modulate motor outputs. dI3 neurons mediate corrective abilities using efferent copies of motor outputs Given that the circuitry is specific amongst spinal cord excitatory neurons in the region of convergence, we next sought to determine whether dI3 neurons play a fundamental role in correcting ongoing movements. To allow for reversible manipulation of dI3 neurons, the expression of the inhibitory DREADD receptor hM4D(Gi) was induced under the control of both Cre and Flp recombinases in Isl1 cre/wt ;vGlut2 flp/wt mice. The expression of the hM4D(Gi) receptor in Isl1 cre/wt ;vGlut2 flp/wt mice was induced either by crossing them with Flp ON -Cre ON -HA-hM4D(Gi) mice to target all dI3 neurons (dI3 broadly-down , Figure 5A , 51 ) or by injecting bilaterally an AAV2/5-hSyn-Flp ON -DIO-HA-hM4D(Gi)-mCherry along the lumbar spinal cord to restrict the manipulation to lumbar dI3 neurons (dI3 lumbar-down , Figure 5B ). We confirmed the reduction of dI3 excitability in the dI3 lumbar-down mice in spinal cord slices, showing that application of the agonist JHU37160 (230nM) led to an increase of ∼80% in cell rheobase, together with a decrease of about 20% in cell input resistance. The agonist did not have an effect on non-transduced, hM4D(Gi) OFF dI3 neurons, confirming that its effect was specific (( Supplemental figure 5A-C ). These results are further supported by our previous experiments where the grasp reflex, a reflex known to be mediated by dI3 neurons 16 , was successfully reduced in neonatal mice using inhibitory DREADD 51 . Download figure Open in new tab Supplemental figure 5: JHU37160 induces a reduction of excitability specifically in dI3 hM4D(Gi) ON while mecamylamine shows no modulation of dorsal root evoked excitatory inputs on dI3 neurons. (A) Step or ramp of current responses from dI3 neurons expressing hM4D(Gi) following injection of AAV2/5-hSyn-Flp ON -DIO-HA-hM4Di-mCherry in the lumbar spinal cord in control condition or after perfusion of JHU37160 (230nM) to the bath. (B-C) Change in (B) cell rheobase and (C) cell resistance in dI3 neurons with (dI3 hM4-mChe) or without (dI3 tdTomato) expression of hM4D(Gi) following perfusion of JHU37160 to the bath. (D) Schematic of the spinal cord preparation that was used to record dorsal root evoked excitation of dI3 neurons. (E) Representative traces of dorsal root evoked excitation in a dI3 neuron in control condition and following addition of mecamylamine to the bath. (F) Amplitude of the dorsal root evoked excitatory responses in control conditions and following addition of mecamylamine (100-200µM). Total of 5 root responses in 4 dI3 neurons from 4 preparation from 3 animals. Non-hierarchically bootstrapped paired samples Wilcoxon tests and paired Hedge’s G. Download figure Open in new tab Figure 5: dI3 neurons mediate corrective responses using efferent copies from Renshaw cells. (A-B) Intersectional strategies used to inhibit reversibly dI3 neurons activity (A) genetically in the whole animal or (B) combining genetic and viral transduction to specifically manipulate lumbar dI3 neurons. (C, F) Schematic of the (C) uneven elevated horizontal ladder and (F) horizontal elevated vibrating beam tasks used to assess corrective responses in mice. (D, G) Averaged number of hindlimb slips per mouse when crossing the elevated (D) ladder or (G) beam. Dots show the values for individual animals with a broad expression of hM4D(Gi) (in black) or an expression restricted to lumbar dI3 neurons (in red). dI3 ON (in green) correspond to session preceded by a subcutaneous injection of saline together with an intraperitoneal injection of saline or mecamylamine (5mg/kg, MEC) while dI3 down sessions (in magenta) were preceded by a subcutaneous injection of the DREADD agonist JHU37160. Values obtained from the same animal are connected with grey lines. (E, H) Matrix of the p values (in red, lower corner, calculated from 2 way repeated measures ANOVA test followed by Tukey’s multiple comparisons test) and absolute effect size (in blue, upper corner, bootstrapped paired Hedge’s G coefficients) obtained between the different groups. Values are indicated in white for significant differences. Since the presence of spinal cord modules combining sensory feedback and efferent copies could theoretically allow for detection of mismatches to generate corrective responses with minimal delays 3 , 10 , we next tested the performance of mice with a reduction of dI3 neuron activity during complex motor tasks. We turned to a behavioural test in which a horizontal ladder had unevenly spaced rungs. In the absence of hM4D(Gi), there were no differences in hindlimb slips when either saline or JHU37160 was given (0.4 ± 0.3 vs 0.3 ± 0.2 slips after saline and JHU37160 administration respectively, n=6 animals). In dI3 broadly-down and dI3 lumbar- down mice, however, the number of hind paw slips following JHU37160 administration increased ( Figure 5C-E ), similar to what was previously described for a horizontal ladder test in a model of global dI3 output suppression 16 . Similar results were seen on an elevated vibrating beam ( Figure 5F-H ). To test whether there could be a role of efference copy from Renshaw cells in the corrective adjustments by dI3 neurons, we ran the same experiment preceded by an intraperitoneal injection of mecamylamine (5mg/kg), a nicotinic antagonist that disrupts Renshaw cell activity by inhibiting their activation by motor neurons ( Figure 2I-K ) but shows no significant effect on sensory inputs to dI3 neurons in longitudinal preparations (( Supplemental figure 5D-F ). In dI3 ON animals, mecamylamine administration led to a significant increase (large effect size) in the number of hindlimb slips compared to saline injected dI3 ON mice in both motor tasks ( Figure 5D-E, G-H ). Conversely, in both dI3 broadly-down and dI3 lumbar-down mice given JHU37160, mecamylamine may have slightly increased the number of hindlimb slips (there was a medium effect size, but p=0.086 in ladder task, 0.35 in beam task), suggesting that the effect observed in dI3 ON animals largely stems from the reduction of Renshaw cell inputs to dI3 neurons ( Figure 5D-E, G-H ). Of note, the absolute number of slips in response to mecamylamine in dI3 ON mice was lower than that in dI3 down mice ( Figure 5D, E, G, H ), suggesting that there may have been an incomplete block of efference copy (from Renshaw cells or another source), or that dI3 neurons also mediate fine corrective movements through integration of tactile perception independently of the efferent copy they receive. In summary, this set of experiments shows that dI3 neurons play a role in corrective motor adjustments during motor tasks. The results seen when inhibiting nicotinic receptors further point to a role for Renshaw cells in the implementation of these corrections. dI3 neurons combine sensory feedback and efference copy to mediate reflexive corrections To determine whether corrective responses depending on motor dynamics are mediated by spinal circuits involving dI3 neurons, we turned to stumbling corrective reactions. Stumbling correction, originally described in cats and present in mice and humans, involves hyperflexion of the knee and the ankle in response to perturbation of the limb during swing phase, leading to stepping over the obstacle that would otherwise have prevented the progression of swing 12 – 14 , 52 – 54 . This reaction (i) is phase dependent, (ii) persists after lower thoracic spinal cord transection 13 , 55 , and (iii) is triggered within a few milliseconds 54 . Furthermore, given these features, this reaction, triggered by a sensory stimulus, (i) needs information about the phase of the step cycle, i.e. an efferent copy, (ii) is generated locally within the lumbar spinal cord circuits, and (iii) involves a minimal number of synapses. These three features led us to address whether the stumbling corrective response could be mediated by dI3 neurons. To trigger a stumbling corrective reaction in mice, we first applied a mechanical perturbation to the dorsal side of the paw during the swing phase of locomotion ( Figure 6A , 14 ). This stimulus resulted in a burst of muscle activity and hyperflexion of the ankle and knee ( Figure 6B-C ). We found that the maximum height of the step during a stumbling corrective reaction (SCR) was reduced during the SCR step in dI3 down mice (both dI3 broadly-down and dI3 lumbar-down ) compared to dI3 ON mice ( Figure 6D , ( Supplemental figure 6B ). Following administration of mecamylamine, there was no difference between the SCR of dI3 ON and dI3 down mice, with a step height similar to the one observed in dI3 down mice injected with saline ( Figure 6D-E , ( Supplemental figure 6B ). These results suggest that dI3 neurons use the efference copy from Renshaw cells to mediate the stumbling corrective response. Download figure Open in new tab Supplemental figure 6: dI3 neurons modulate stumbling correction combining sensory feedback and efference copy from Renshaw cells. (A-F) Matrix of the p values (in red, bottom corner, calculated from 2 way repeated measures ANOVA test followed by Tukey’s multiple comparisons test) and absolute effect size (in blue, top corner, bootstrapped paired Hedge’s G coefficients) obtained while comparing step height between the different groups for (A-C) mechanical and (D-F) electrical perturbations in the step before, during and after SCR. Values are indicated in white when differences are significant. Download figure Open in new tab Figure 6: dI3 neurons modulate stumbling correction combining sensory feedback and efference copy from Renshaw cells. (A, F) A stumbling corrective reaction (SCR) was triggered using (A) a mechanical perturbation or (F) an electrical stimulation of the saphenous nerve. (B, G) Raw EMG of the tibialis anterior showing the step cycle before, during and after (B) mechanical or (G) electrical perturbation of the swing. The perturbation is indicated by the grey inside and arrow on the EMG of the second swing. (C, H) Stick diagram reconstruction of a swing during the SCR with or without inhibition of dI3 neurons and with or without inhibition of cholinergic transmission using mecamylamine (5mg/kg). The SCR is triggered by (C) mechanical or (F) electrical perturbation, the moment of the perturbation is shown in red and the trajectory of the toes in blue. (D, E) Step height of the step before, during and after mechanical SCR following intraperitoneal injection of (D) saline or (E) mecamylamine in animals without (dI3 ON , green lines) or with an inhibition of dI3 neurons (dI3 down , magenta lines). Lines connect averaged step height of the same animal, while dots represent averaged values of each animal for broad dI3 silencing (in black) and inhibition restricted to lumbar dI3 neurons (in red). (I, J) Step height of the step before, during and after electrical SCR following intraperitoneal injection of (I) saline or (J) mecamylamine in animals without (dI3 ON , green lines) or with an inhibition of dI3 neurons (dI3 down , magenta lines). Lines connect averaged step height of the same animal, while dots represent averaged values of each animal for broad dI3 silencing (in black) and inhibition restricted to lumbar dI3 neurons (in red). (D, E, I, J) 2 way repeated measures ANOVA test followed by Tukey’s multiple comparisons test, only significant differences are indicated. However, in these experiments there was an increase in the height of the step preceding that with the mechanical perturbation in the dI3 ON animals injected with saline, raising the possibility that an anticipatory response interfered with the results ( Figure 6D , ( Supplemental figure 6A ). To circumvent this concern, we next induced a stumbling corrective response using electrical stimulation of the saphenous nerve during the early swing phase ( Figure 6F ). This stimulation reliably triggered a stumbling corrective reaction similar to that described previously 14 , with hyperflexion of the homonymous limb and a burst of activation in the ankle flexor ( Figure 6G-H ). In these experiments, the step height of the step before stimulation was similar in all the different group ( Figure 6I-J , ( Supplemental figure 6D ). During SCR, we observed, a decreased amplitude of the corrective reaction in both dI3 broadly-down and dI3 lumbar-down models ( Figure 6I , ( Supplemental figure 6E ). Furthermore, mecamylamine administration in dI3 ON animals led to SCR step heights similar to those seen in dI3 broadly-down and dI3 lumbar-down models ( Figure 6I-J , ( Supplemental figure 6E ). Step heights during SCR following mecamylamine administration in dI3 ON animals were similar to those in dI3 broadly-down and dI3 lumbar-down mice following mecamylamine administration ( Figure 6J , ( Supplemental figure 6E ). We therefore conclude that the reduction of the stumbling corrective reaction observed when dI3 ON animals were administered mecamylamine was due to the disruption of inputs to dI3 neurons, suggesting a pivotal role of Renshaw cell inputs to dI3 neurons for the generation of reflexive corrections. Lastly, in the step following both types of perturbation – mechanical or electrical – we detected a different height between dI3 ON animals without mecamylamine injection and all other groups, suggesting a potential role of this circuit in modulation over the longer term (( Supplemental figure 6C, F ). Taken together, our results describe spinal circuits in which dI3 neurons combine the integration of sensory feedback and efferent copies of motor outputs via Renshaw cells and motor neurons. We further show that this circuit is responsible for mediating correction of ongoing movements. These data point to spinal motor circuits that use predictive inputs from efference copies to generate rapid corrections and adjustments of ongoing movements. Discussion In this study our aim was to determine whether spinal dI3 neurons could form comparator modules for fast correction of ongoing movements. Using circuit tracing, we describe the convergence of multimodal sensory inputs and internal motor copies, including from Renshaw cells, on dI3 neurons. We show that these instructive and predictive inputs converge on individual dI3 neurons. In addition, dI3 neurons innervate motor neurons favouring flexor over extensor and for a subpart forming divergent connections to antagonist sets of motor pools. By manipulating their activity during behaviour, we demonstrate the pivotal role of dI3 neurons in corrective responses. We further show that nicotinic transmission is necessary for the generation of corrective dI3-dependent responses, suggesting that the efference copy from Renshaw cells on dI3 neurons is essential for the generation of fast corrections of movement. Based on this converging evidence, we thus propose that dI3 neurons form comparator modules in the spinal cord ( Figure 7 ). Download figure Open in new tab Figure 7: dI3 neurons combine sensory feedback and internal motor copies for corrections of ongoing movements. In an intact circuits dI3 neurons combine multimodal sensory feedback and internal motor copies from Renshaw cells to induce a fast correction of movements following a perturbation. When dI3 neurons or Renshaw cells activity are removed from spinal circuits fast corrections are abolished. dI3 neurons receive segregated, instructive inputs from multiple modalities dI3 neurons are distributed along the medio-lateral axis in the intermediate laminae (Supp Figure 1A-C , 16 ). Further, their position differs along the rostro-caudal axis even within the lumbar spinal cord: for instance, the most medial dI3 neurons are mainly in the caudal segments of the lumbar cord (Supp Figure 1D ). On this anatomical basis alone, it would therefore seem that this cardinal class, like others 56 , 57 , could be formed by several subclasses which may differ molecularly, morphologically, and by their inputs, and thus may serve distinct functions. It is clear that the position and the dendritic arborization of neurons are crucial to specify the inputs they receive 19 , 57 – 59 . We found that a large subclass of dI3 neurons located in an interposed position along the medio-lateral axis is distinct from others and likely comprise several sub-populations. This position overlaps with the “region of convergence” of proprioceptive axons from multiple muscles 19 , and these interposed dI3 neuronal somata are indeed densely innervated by these afferents ( Figure 1A-E ; Supp Figure 1E-G ). Moreover, we found that cutaneous inputs are segregated to their dorsal dendritic branches ( Figure 1F-I ). Thus the morphology and position of dI3 neurons may be critical to their function. This modality-dependent segregation of inputs across somato-dendritic compartments may have important roles in the modulation of gain, modality-dependent integration of sensory feedback, and plasticity. It has been shown that the segregation of inputs to the distal dorsal dendrites of layer 5 pyramidal neurons allows for an increase in gain of tactile feedback during active perceptual detection 60 , 61 . Similarly, the spatial segregation of cutaneous inputs to dI3 neuronal dendrites may provide the substrate for specific modulation of these inputs and may explain, for instance, how descending monoaminergic inputs can modulate cutaneous reflexes 62 . Dendritic plateau potentials could also provide a mechanism for non-linear interactions across modalities 63 – 65 . Localizing cutaneous inputs to the dendrites may also provide a mechanism for specifically gating them. Although we did not investigate the spatial distribution of inhibitory inputs along the somato-dendritic compartment their distribution can impact their function 66 , 67 . For instance, inhibitory inputs received by dI3 neurons following sensory afferent stimulation may reduce the effects of cutaneous feedback to the dorsal dendrites, independently of other inputs 68 . This compartmental segregation may allow more flexibility for the regulation of synaptic plasticity. Indeed, as observed in hippocampal microcircuits, local cooperativity of inputs of the same modality may modulate plasticity locally 69 , 70 , having no plastic impact on other sensory modalities. Given the fundamental role of dI3 neurons in plasticity of spinal circuits following complete transection 17 , these observations open new directions to study further the role of somato-dendritic compartmentalization in the integration of feedback and feedforward signals in spinal circuits. Renshaw cells provide predictive inputs to dI3 neurons While some basic spinal reflexes rely largely on sensory feedback 25 , 27 , experiments on different species such as, mice, cats, and humans have shown that corrections following sensory perturbations are dependent on the phase of ongoing movement such as the locomotor cycle 12 – 14 , 52 . To do this, the interneurons mediating these corrective actions must have some “knowledge” of the motor activity; such inputs are provided by efference copies of the motor commands. We have previously showed that dI3 neurons receive rhythmic inputs from spinal locomotor circuits 17 . Although we could not identify the source of these inputs, their phase-dependence led us to suggest that dI3 neurons receive an efference copy. Here, we show that dI3 neurons modulate phase-dependent corrections of movements, and that one possible source of locomotor efference copies may arise from Renshaw cells. Renshaw cell activity arguably represents the most direct and accurate negative efferent copy of motor activity 10 , as they are activated by motor neuron collaterals 71 , 72 through a ‘relay’ synapse 73 . Since a negative efferent copy is pivotal in mismatch detection between excitatory sensory feedback and motor commands, following our observation of the inhibition received by dI3 neurons from efferent copies, we sought to acutely disrupt their activity. Previously, a genetic strategy was used to manipulate Renshaw cell activity and suggested that they may play a role in long term plasticity of spinal circuits 74 . However, this strategy is not specific to Renshaw cells 36 , 74 and could not be used together with a manipulation of dI3 neurons. We therefore, opted to use a pharmacological approach 39 , 75 , 76 to disrupt motor copies received by Renshaw cells. The inhibition of the response to perturbation during the swing phase by mecamylamine demonstrated that nicotinic transmission is involved in stumbling corrections. Although we cannot exclude a concomitant effect on the modulation of other pathways, including primary afferents 77 , 78 , the evidence supporting that the effects were mediated via Renshaw cells includes: (a) the synaptic connectivity, with Renshaw cells connecting to dI3 neurons ( Figure 2A-H , ( Supplemental figure 2A-D ); (b) the reduction in Renshaw cell activity with mecamylamine resulting in decreased inhibition of dI3 neurons ( Figure 2I-M ); (c) the minimal effects of mecamylamine on afferent transmission to dI3 neurons (( Supplemental figure 5D-F ); and (d) the use of this strategy in fictive preparations, to show a reduction in Renshaw cell activation that resulted in reduced inhibition of motor neurons 39 , 75 . Thus the preponderance of evidence points to Renshaw cell involvement. How could a reduction in predictive, inhibitory, input lead to a smaller corrective response? Comparators use internal representations to compare to instructive external signals – the removal of any inputs could thus lead to a failure in corrections. The role of Renshaw cell inputs to motor neurons has long been debated, with suggestions including, for example, desynchronisation of motor neuron firing or changing the recruitment gain of the whole pool 79 . Alternatively, the reduction of corrections might be explained by the role of Renshaw cell inputs in coordinating dI3 neuronal activity, for instance through post-inhibitory rebound 80 . In addition, the segregation of inputs to dI3 neurons that project to flexor vs extensor motor neurons is not known. Renshaw cells are known to inhibit the motor pool that activates them whilst disinhibiting antagonist pools via Ia inhibitory interneurons 81 . If there is a similar organisation with respect to dI3 neurons, it is possible that, in response to a stumbling stimulus, they inhibit extensor-related dI3s and disinhibit flexor-related dI3s, in which case mecamylamine would reduce the corrective response. It is also interesting to note that inputs from Renshaw cells would necessarily precede instructive feedback and may therefore favour timely rebound activity upon excitatory feedback 80 , leading to increased flexion. That is, there are several possible circuits and cellular mechanisms through which Renshaw cell input to comparators can contribute to corrective responses. Here, we suggest that Renshaw cells are a source of predictive information within spinal circuits, but predictive inputs are likely not restricted to this pathway. For instance, dI3 neurons – at least in the cervical spinal cord - also receive inputs from corticospinal neurons 51 , 82 . Descending inputs may modulate the corrective signals generated by dI3 neurons as has been suggested for spinal RORα neurons 26 . In fact, the effect of descending regulation of dI3 neurons may be reflected in the anticipatory response observed in the step prior to stumbling corrective reactions triggered by mechanical perturbation: the step prior to the perturbation was significantly reduced when dI3 neurons were downregulated. Therefore, dI3 neurons may additionally process descending inputs to anticipate potential changes in the environment, and trigger anticipatory modulation of locomotion. A spinal comparator module Comparison between internal copies and instructive feedback occur in several brain circuits; and have been extensively studied in cerebellar systems and in the neocortex 9 , 83 , 84 . The location of a comparator module in spinal circuits leads to an advantage in term of delays, fundamental to trigger immediate corrections and prevent severe consequences. However, predictive processing in spinal circuits likely differs from the multifactorial tuning present in higher brain regions. Therefore, in addition to the generation of immediate corrections, dI3 neurons may relay these errors to higher centres 85 , to further refine corrections across longer time scales. Indeed, although corrections occur at different timescales and with different roles, circuits involved in the generation of predictive processing are not working in isolation. For instance, cerebellar outputs modulate neocortical predictive processing 83 , while climbing fibre activity associated with withdrawal reflexes may update cerebellar predictions following immediate corrections 86 . Therefore, in the future, it will be important to characterise how spinal reflexive corrections are used to update predictive models in higher hierarchical levels. Limitations In this study, we identified the nature of the instructive and predictive inputs on dI3 neurons ( Figure 1 and 2 ). We further showed that dI3 neurons receive proprioceptive signals and project to motor neuron pools of both extensor and flexor muscles ( Figure 3 and 4 ). Our interpretation is tempered by two main limitations. Firstly, our pharmacologic manipulation of Renshaw cells may have affected other, relevant systems (see above). And secondly, we did not determine the specificity of circuits involving flexor vs extensor (vs both) projecting dI3 neurons. The future description of the organisation of flexor versus extensor related excitatory and inhibitory inputs and their patterns of divergence versus convergence to dI3 neurons will help to refine our understanding of how dI3 neurons generate immediate corrections during motor behaviour. Methods Animals All experiments were performed according to the Animals (Scientific Procedures) Act UK (1986) and certified by the UCL AWERB committee, under project license number PP2688499 or under the approval of university of Ottawa’s animal care committee and conform to the guidelines put forth by the Canadian Council for Animal Care. Heterozygous Isl1 cre/wt (Jackson Lab, stock #024242) were crossed with homozygous Ai14 fl/fl (Jackson Lab, stock #007914) or heterozygous Ai34d fl/wt (Jackson Lab, stock #012570) to obtain respectively a somatic expression of tdTomato or its expression at presynaptic terminals. The same line was crossed with Slc17a6 -IRES2-FlpO-D mice (referred as vglut2 flp/wt , Jackson Lab, stock #030212) to allow the reduction of excitability in lumbar dI3 neurons and further crossed with Cre ON -Flp ON -hM4D(Gi) (Jackson Lab, stock #029040) to target all dI3 neurons. Heterozygous En1 cre/wt (Jackson Lab, stock # 007916) were crossed with heterozygous Ai34d fl/wt to induce tdTomato expression at the presynaptic compartment of V1 neurons. For experiments aimed at recording from Renshaw cells, heterozygous Slc6A5e GFP/wt mice (termed glyT2-eGFP here, a gift from Prof. Zeilhofer, University of Zurich, 87 ) were used and further crossed with Isl1 cre/wt Ai14 fl/wt to identify non-dI3 excitatory neurons. Homozygous Chat cre/cre mice (Jackson lab, stock #006410) were crossed with homozygous Rosa26 R Φ GT mice (Jackson Lab, stock #024708), to generate Chat cre/wt ; Rosa26 R Φ GT that were used for rabies tracing experiments 47 , 49 , 88 . All mice used were in a C57BL6/J genetic background. For behavioural experiments, all mice were housed individually in a reverse light-cycle room with 12 hours of light from 7pm to 7am; all behavioural testing was performed in simulated darkness using red LED and infrared lighting between 8am and 5pm. Modified rabies virus production The glycoprotein G-deleted variant of the SAD-B19 vaccine strain rabies virus was used (kind gift from Dr M.Tripodi). Modified RabV (ΔG-RV) with the sequence of the glycoprotein G replaced by the mCherry or eGFP sequence (ΔG-RV-eGFP/mCherry) was produced at a high concentration with minor modifications to the original protocol 89 . BHK cells expressing the rabies glycoprotein G (BHK-G cells) were plated in standard Dulbecco modified medium supplemented by 10 % foetal bovine serum (FBS) and split after 6–7hr incubating at 37°C and 5 % CO2. The cells were then inoculated at a multiplicity of infection of 0.2–0.3 with either ΔG-RV-eGFP or mCherry virus (initial samples kindly provided by Prof. Arber and Dr. Tripodi). Cells were incubated for 6 hours at 35°C and 3% CO 2 and subsequently split 1 to 4 with 10% FBS medium and kept at 37°C and 5% CO 2 for 12-24 hours. The medium was then replaced with 2% FBS supplemented medium and cells were incubated at 35°C and 3% CO 2 for virus production. The supernatant was collected after ∼3 days and new medium was added for another round of production (three cycles maximum). The supernatant was filtered (0.45 μm filter) and centrifuged for 2 hours at 19 400 rpm (SW28 Beckman rotor). The pellets were suspended in PBS, dispersed and collected in a single tube and further centrifuged for 4 hours at 21 000 rpm in a 20% sucrose gradient (SW55 Beckman rotor). The resulting pellet was suspended in 100 μl PBS and the virus was stored at −80°C. The viral titre of each round of production was measured by serial 10-fold dilution of three different aliquots using standard protocols. Intramuscular injections Neonatal pups (P2 for ΔG-RV injections, P7-8 for CTB injections) were anaesthetized using isoflurane inhalation and the skin above the injection site was wiped with ethanol 70%. For hindlimb injections, an incision was made on the skin to visualize the targeted muscle either tibialis anterior (TA), lateral gastrocnemius (LG, whose primary role is that of ankle extensors, but also contribute to knee flexion) or gastrocnemius (GS) without distinction between lateral and medial when injecting CTB. Each muscle was injected with 1 µL of ΔG-RV or 0.5µL of CTB coupled to Alexa 488 (ThermoFisher, C34775) or 647 (ThermoFisher, C34778). Subcutaneous injections in the hind paw were performed without incisions, using 0.5µL of CTB-Alexa 488 or Alexa 647. The injected ΔG-RVs were used at a titre between 10 9 and 10 10 IU/ml while CTB was used at 0.5%. All the injections were performed using a 5 μl Hamilton syringe (model 7652-01) fixed to a manual Narishige micromanipulator (M-3333) and loaded with a bevelled (30°) glass pipette of outer diameter 40-45 μm (1B100-4, World Precision Instrument). After slowly injecting the virus (> 1 minute), the needle was left on the injection site 2-3 minutes and then retracted. In the following 24 hours, injected pups were closely monitored to control for signs of movement impairment or rejection by the mother. The injected pups were perfused under terminal general anaesthesia respectively 9 days (ΔG-RV) and 3-4 days (CTB) after injections. Intraspinal injections AAVs to visualize dI3 somato-dendritic compartment were injected in P15-P16 animals while 5-7 weeks old animals were injected to allow acute manipulation of lumbar dI3 neurons. Animals were first given Buprenorphine (Buprecare, XVD 130) to induce analgesia and then put under general anaesthesia with isoflurane (5% for induction, 1-1.5% maintenance). To stabilise the spinal cord of young pups a custom device for stabilisation of the vertebrae was used while adult animals were placed onto a stereotaxic frame (Kopf Instruments, Model 963 Ultra Precise Small Animal Stereotaxic Instrument). Intraspinal injections for anatomy and electrophysiology experiments were performed using a pulled and bevelled borosilicate glass pipette (Warner instruments, G120F-4) with an outer diameter of 25-35µm at the tip, attached to a stereotaxic device (Narishige, SMM-200). AAVs were injected applying multiple short pulses using a pneumatic picopump (World Precision Instruments, PV820). For behavioural experiments, injections were performed using a pulled and bevelled quartz capillary (Sutter Instruments, Q100-70-7.5) with tip at an outer diameter of 25-45 µm, attached to a Hamilton syringe (Hamilton, CAL7632-01) via a compression fitting (Hamilton, 55750-01), and mounted to the stereotaxic frame on a microinjector (Harvard Apparatus, Pump 11 Elite, Cat.# 70-4507). To label dI3 neurons prior to retrograde tracing from the sural nerve, a dorsal laminectomy of T13 was performed and 5 sites were injected on one side of the cord from L3 to caudal L5. For each site, 80 to 120nl of AAV1-phSyn1-flex-Lyn/EYFP-T2A-synaptophysin:mKate2 (VectorBuilder, ID VB180425-1066vcf, only the EYFP signal targeted by the Lyn domain to the membrane was used, abbreviated AAV1-phSyn1-flex-mYFP) was injected in the intermediate laminae (350µm medio-lateraly, 650µm dorso-ventrally) three weeks before tissue collection. To induce the expression of the DREADD receptor hM4D(Gi) specifically in lumbar dI3 neurons, mice were injected with an AAV2/5-hSyn-Flp ON -DIO-HA-hM4D(Gi)-mCherry (final titer of 4 × 10 12 vp/mL, 2064-aav5, Canadian Neurophotonics Platform Viral Vector Core Facility, RRID:SCR_016477). The intermediate laminae were injected on three sites on each side of the cord, 250-300nl per site, using the laminar spaces between T11-T12 (300µm medio-lateraly, 670µm dorso-ventrally), T12-T13 (350µm medio-lateraly, 700µm dorso-ventrally) and T13-L1 (350µm medio-lateraly, 650µm dorso-ventrally). To control the Cre dependence of each AAV construct, Cre negative animals were injected in the lumbar cord similarly to Cre positive animals, in at least 2 animals per construct and no fluorescent cells were observed at the sites of injection even after amplification of the fluorescent protein with immunoreaction. To control the specific expression of double Cre and FlpO dependant AAV2/5-hSyn-Flp ON -DIO-HA-hM4D(Gi), the construct was injected in Cre ON -Flp OFF and Cre OFF -Flp ON animals. A maximum of up to 5 cells were labelled in the sections adjacent to the injection sites (45 μm thickness), with none detected further than 90 μm from the injection site. These represent a negligible proportion of labelled cells in Cre ON -Flp ON animals used for behavioural experiments Retrograde labelling from the sural nerve To label the cutaneous sensory axons from the sural nerve, mice were anaesthetized using isoflurane following induction of analgesia. An incision was made above the distal limb and the nerve was isolated from the facia and cut. The nerve was then put on a parafilm, surrounded by vaseline and dipped into a solution of CTB-Alexa 647 at 0.5% (Thermo Fisher Scientific, C34778) for 1 hour. The extremity of the nerve was washed multiple times with saline and the overlying skin was sutured without reattachment of the extremity of the nerve. The animals were then perfused five days after the procedure. After perfusion, the dorsal root ganglions of L3 to L6 were collected and stained for parvalbumin and CTB to verify the identity of sensory neurons labelled. Slices selected randomly were imaged, and 8/197 (n=2 animals) sensory neurons quantified expressed parvalbumin, confirming that the vast majority of sensory boutons traced were from cutaneous sensory neurons. Patch-clamp electrophysiology Spinal cord preparations and solutions Mice were first anaesthetized with a mixture of ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively) and decapitated. The spinal column was dissected and a ventral laminectomy was performed in a chamber filled with cold (∼2°C) artificial cerebrospinal fluid (aCSF, the same used for recordings) composed of (in mM): 113 NaCl, 3 KCl, 25 NaHCO 3 , 1 NaH 2 PO 4 , 2 CaCl 2 , 2 MgCl 2 , and 11 D-glucose continuously bubbled with 95% O 2 and 5% CO 2 . To map the functional inputs to dI3 neurons several slicing approaches were used to stimulate ventral and/or dorsal L4-L5 roots while recording from dI3 neurons, motor neurons or Renshaw cells. After laminectomy, the lumbosacral cord was glued to an agar block (7% agar/0.1% methyl blue) either ventral or dorsal horn facing up for experiments requiring ventral and dorsal-root stimulations, respectively. The tissue was then placed in a chamber filled with ice-cold slicing aCSF (∼2°C) comprising (in mM): 130 K-gluconate, 15 KCl, 0.05 EGTA, 20 HEPES, 25 d-glucose, 3 Na-kynurenic acid, 2 Na-pyruvate, 3 Myo-inositol, 1 Na-l-ascorbate, pH 7.4 with NaOH 90 and sliced longitudinally 91 or with an angle (∼45°, 350 µm thickness) using a vibratome (HM 650 V, Microm, Thermo Fisher Scientific). Slice(s) were incubated in a chamber with recording aCSF (constantly bubbled with 95/5% O 2 /CO 2 ) at 37°C for about 30min, then maintained and recorded at room temperature (∼22°C). For hemisected longitudinal preparation, we did not slice the cord using a vibratome, but instead carefully hemisected the cord in recording aCSF before performing the recordings. To investigate synaptic inputs in response to ventral or dorsal root stimulation (see 91 for further details) in voltage-clamp mode, a caesium-based intracellular solution containing QX-314 to block spikes was used. This intracellular solution included (in mM): 125 Cs-gluconate, 4 NaCl, 0.5 CaCl 2 , 5 EGTA, 10 HEPES, 3 QX-314-Br, 2 Mg-ATP, pH 7.3 with CsOH, and osmolarity of 290–310 mosmol. Inhibition and excitation were recorded at their calculated equilibrium potential (+15 and −60 mV, taking into account the calculated junction potential of ∼-15 mV for all the intracellular solutions we used).To assess the inhibitory effect of DREADD agonist JHU37160, we used GTP-enriched, ATP-regenerating intracellular solution containing (in mM): 140 K-gluconate, 10 KCl, 0.1 EGTA, 10 HEPES, 2 Mg-ATP, 0.5 5′-GTP-Na 2 , 5 phosphocreatine-Na 2 , pH 7.3 with KOH, and osmolarity of 290–310 mosmol. For loose-cell attached recordings the electrode was filled with recording aCSF. Recording configurations and analysis of responses Ventral- or dorsal-horn ablated longitudinal spinal cord preparations from Isl1 cre/wt ;Ai14 fl/wt were used to infer about excitatory and inhibitory drive from sensory afferent or motor efferent activation to dI3 neurons. We used borosilicate glass pipettes (∼1.5x the root diameter) to stimulate dorsal or ventral L4 and/or L5 roots. We initially patched a motor neuron to define the stimulus threshold, i.e. the minimum stimulus intensity that generates a clear excitatory synaptic response, and then used 2-3x threshold intensity for the rest of the experiment. Following root stimulations (an average of 10 sweeps with interstimulus interval of ∼10 seconds and stimulus duration of 0.1-0.2 ms), the stimulus artifact was subtracted from the recording trace with a single or double exponential, and baseline-to-peak amplitude (size) and the artifact-to-first response time (latency) for each peak were calculated. The conductances (σ) of the root-evoked excitatory (EPSC) or inhibitory postsynaptic currents (IPSC) measured in voltage clamp, were calculated at the holding voltage taking into consideration a reversal of 0 mV for excitatory and −60 mV for inhibitory conductances. In the case of multipeaked responses, we calculated the size and the latency of the first peak. The amplitude and latency of the EPSCs and IPSCs were averaged across sweeps, whereas the jitter was calculated as the variance of the latency for all sweeps. For dI3 neurons which responded to root stimulations, cell coordinates along mediolateral axis were taken. In these set of experiments, we also injected a 500ms-long current to calculate i) the membrane time constant, ii) cell conductance and iii) capacitance in current clamp mode 92 . In the subset of dorsal-horn ablated preparations (on the last cell patched in the preparation), stimulation of the ventral root was repeated after addition of 3 µM 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) and 50 µM 2-amino-5-phosphonopentanoic acid (APV or AP5) to block AMPA and NMDA receptors respectively. Recordings of Renshaw cells were performed in oblique slices obtained from glyT2-eGFP animals. First, we recorded from a lumbar motor neuron to set the stimulation intensity for ventral root stimulation as previously described. Next, we identified Renshaw cells based on their i) location (laminas VII and IX), ii) expression of eGFP and iii) presence of extracellular spikes in response to ventral root stimulation in loose-cell attached mode. We performed whole-cell recordings from motor neurons and loose-cell-attached recordings from Renshaw cells, to measure ventral root-evoked extracellular spikes in Renshaw cells and IPSCs in motor neurons over a minimum of 10 sweeps. We then perfused 100µM mecamylamine (Sigma, M9020) for at least 15 minutes. Additionally, in spinal cord slices obtained from Isl1 cre/wt ;Ai14 fl/wt mice, we recorded ventral root-evoked inhibitory and dorsal root-evoked excitatory responses from dI3 neurons before and after application of 100 µM mecamylamine. Hemisected preparations from In Isl1 cre/wt ;Ai14 fl/wt animals were used to measure ventral and dorsal root responses from dI3 neurons. We stimulated ventral and dorsal roots (L4 or L5 roots) with the stimulation intensity set as 2-3x the threshold required to elicit an initial synaptic response in dI3 cells measured in whole-cell mode. In Isl1 cre/wt ;Ai14 fl/wt ;glyT2-eGFP animals, we conducted the same experiments to additionally record from non-dI3 excitatory neurons. We first performed patch-clamp recordings from dI3 neurons to determine the threshold stimulation intensity and confirm the quality of the preparation, and then we targeted eGFP OFF /tdTomato OFF , i.e. non-dI3 excitatory neuron. For these sets of experiments, we excluded 2 hemisected preparations in which there were no cells showing a response to stimulation of the dorsal root, suggesting that most of the sensory axons had been damaged. To validate the effect of the DREADD receptor agonist JHU37160 on silencing dI3 neurons expressing hM4D(Gi), we performed whole-cell recordings from dI3 neurons in oblique spinal cord slices from adult Isl1 cre/wt ;vglut2 flp/wt mice. These mice were injected with AAV2/5-hSyn-Flp ON -DIO-HA-hM4D(Gi)-mCherry and age-matched to those used in behavioural experiments. To assess the specificity of JHU37160, we also recorded from dI3 neurons in non-injected adult Isl1 cre/wt ;Ai14 fl/wt mice. We estimated cell input resistance using a 500ms-long current step and determined rheobase as the minimum current eliciting a spike during the ascending phase of a triangular ramp protocol (4 s up, 4 s down). These measurements were conducted before and after applying 230 nM JHU37160, repeated every 5 minutes for at least 15 minutes. Equipment Cells were observed using a Nikon Eclipse E600FN microscope (Nikon, Japan) equipped with a dual-port system for simultaneous imaging of infrared differential interference contrast (DIC) via a digital camera (Moment-mono-OC) and fluorescence. Excitation was provided by a 488nm LED for EGFP expressing cells, and a 560nm LED for cells expressing TdTomato (Opto LED, Cairns Instruments, UK), and emission was captured using a CCD camera (Retiga XR, QImaging, UK). Whole-cell recordings were performed using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale), loose-cell attached recordings were done using an ELC-03X amplifier (NPI Electronics), and signals were filtered at 5 kHz and acquired at 50 kHz using a Digidata 1440 A A/D board (Molecular Devices, Sunnyvale) and acquired using Clampe× 10 software (Molecular Devices, Sunnyvale). Patch pipettes were pulled from borosilicate glass (GC150F, Harvard Apparatus, Cambridge, UK) with a Flaming-Brown puller (P1000, Sutter Instruments, CA) to a resistance of ∼5 MΩ for dI3 neuron whole-cell and Renshaw cell loose-cell attached recordings, and ∼2 MΩ electrodes were used for motor neuron recordings. The roots were tightly sealed with glass micropipettes cut to similar diameters of L4 and L5 roots, and then used to stimulate the axons through an isolated constant current stimulator (DS3, Digitimer, Welwyn Garden City, UK). EMG electrode and nerve cuff fabrication To record muscle activity and electrically stimulate afferent fibres in the hindlimb during treadmill walking, custom EMG muscle and nerve cuff electrodes were made following the protocols described in previous mouse studies 14 , 93 , 94 . Briefly, for muscle implant electrodes, stainless steel wire (AM System, #793200) was inserted into the head piece connector (Samtec, #CLP-108-02-L-D) and attached to a 27G needle on the remaining end. The nerve cuff electrode was fabricated with the same stainless steel wire, connected to the same head piece while the end of the electrode wires was inserted through a silicone cuff (Implantech, PAT02) to be slipped around the saphenous nerve and tied shut with a silk suture running through the silicone cuff (Ethicon BV-1, K809H). The plastic housing of the connector was then coated in a thin layer of biocompatible gel Epoxy (opaque 5-Minute Epoxy Gel, Devcon 14265). After allowing the Epoxy coating to cure completely for at least 24 hours, implants were then sterilized prior to surgery in 3% hydrogen peroxide solution for 20 minutes followed by a sterile water wash to remove any residual hydrogen peroxide. Nerve cuff and EMG electrode implantation Following recovery after intraspinal injection, surgical implantation of EMG electrodes and nerve cuff was performed. Animals were first given Buprenorphine to induce analgesia and then put under general anaesthesia with isoflurane (5% for induction, 1-1.5% maintenance). EMG electrodes were implanted into the tibialis anterior and lateral gastrocnemius, of both right and left hindlimbs. Additionally, one nerve stimulation cuff electrode was placed around the saphenous nerve of the left hindlimb. Electrodes were implanted following the surgical procedures previously described 94 , 95 . Briefly, incisions were made in the upper neck region and in the skin of the hindlimbs to expose the target muscles and saphenous nerve. The electrodes were drawn through the neck incision down to the target muscle or nerve regions and inserted into the muscle parallel to the fibres or placed around the saphenous nerve. A head piece connector was secured into the skin of the dorsal neck region to allow stimulation from external equipment. Mice were given at least 1 week to recover before any acclimation or training sessions began prior to behavioural testing. Tissue collection and immunohistochemistry The mice were perfused with PBS (0.1 M) followed by PBS 4 % paraformaldehyde (PFA, ThermoFisher, 28908) under terminal ketamine/xylazine anaesthesia (i.p. 80 and 10 mg/kg, respectively). Spinal cords were collected and post-fixed for 2-3 hr in 4% PFA, cryoprotected in 30 % sucrose in 0.1M PBS, embedded in PolyFreeze (Polysciences, 25113-1) or OCT (Tissue-Tek, 4583) and sliced with a cryostat (Leica, CM3050 S). To visualize cutaneous boutons from the sural nerve and dI3 neurons, 100 µm thick free-floating sections were first blocked 30 minutes in PBS 2-X (0.2 M), 0.2-0.3 % Triton 100-X, 10% donkey normal serum, and then incubated with primary antibodies for 72 hours at 4°C and with secondary antibodies 24h at 4°C in the same blocking solution. To quantify the number of dI3 presynaptic boutons onto motor neurons, 50 µm thick sections on slides were first treated with citrate buffer 0.3% (ThermoFisher, 036439.A3), pH 6, at 80°C for 3 minutes. Then sections were blocked 30 minutes in PBS 2-X (0.2 M), 0.2 % Triton 100-X (Sigma, T9284), 10 % donkey normal serum (Sigma, D9663), and incubated with primary antibodies for 72 hr at 4°C and with secondary antibodies 24 hr at 4°C in the same blocking solution. For all the other immunohistochemistry, sections on slides (30-40 µm thickness) were first blocked 30 minutes in PBS 2-X (0.2 M), 0.2-0.3 % Triton 100-X, 10 % donkey normal serum, and then incubated with primary antibodies for 32-36 hours at 4°C and with secondary antibodies overnight at 4°C in the same blocking solution. To assess the presence of homonymous/heteronymous proprioceptive boutons onto premotor dI3 neurons following premotor tracing with RV, sequential immunoreaction and imaging were performed. The first reaction was performed on GFP, mCherry, parvalbumin (PV) and Isl1 as described above. Then, the tissues were imaged, the slides unmounted and incubated with primary antibody against vGlut1 and PV for 24 hr followed by an incubation overnight at 4°C with secondary antibodies. The primary antibodies used were: goat anti-choline acetyltransferase (ChAT, 1:100, Millipore, AB144P), rabbit anti-CTB (1:400, Abcam, ab34992), goat anti-CTB (1:5000, List Labs, 703), chicken anti-mCherry (1:2000, Abcam, Ab205402), rat anti-RFP (1:1000, Chromotek, clone 5F8), chicken anti-GFP (1:1000, Abcam, Ab13970), rabbit anti-GFP (1:2500, Abcam, Ab290), goat anti-GFP (1:500, Abcam, ab6673), guinea pig anti-GFP (1:750, Synaptic systems, 132 005), rabbit anti-Calbindin (1:4000, Swant, CB38a), rabbit anti-PV (from 1:1000 to 1:2000, Swant, PV27a), guinea pig anti-vGlut1 (1:2000, Millipore, AB5905), guinea pig anti-vGlut2 (1:2000, Millipore, AB2251-I) and guinea pig anti-Isl1 (1:7500, RRID:AB_2801512 from Susan Brenner-Morton, Columbia University, New York); and the secondary antibodies: donkey anti-goat Alexa 647 (1:1000, ThermoFisher, A-21447), donkey anti-goat Alexa 488 (1:1000, ThermoFisher, A11055), donkey anti-goat preabsorbed Alexa 405 (1:200, Abcam, Ab175665), donkey anti-guinea pig Alexa 647 (1:700, Millipore, AP193SA6 and 1:1000, Jackson ImmunoResearch, 706-605-148), donkey anti-guinea pig Alexa 488 (1:500, Jackson ImmunoResearch, 706-545-148), donkey anti-guinea pig DyLight 405 (1:400, Jackson ImmunoResearch, 706-475-148), donkey anti-rabbit Alexa 488 (1:1000, Thermo Fisher, A21206), donkey anti-rabbit Alexa 405 (1:500, Abcam, 175649), donkey anti-chicken Cy3 (1:1000, Jackson ImmunoResearch, #703-165-155), donkey anti-chicken CF 488A (1:500, Sigma, SAB4600031), donkey anti-rat Alexa 555 (1:500 to 1:1000, Thermo Fisher, A78945). In some experiments, Neurotrace Blue (1:200, ThermoFisher, N21479) was used together with the secondary antibodies to visualize the shape of neurons somata. Free floating sections were mounted in Ce3D tissue clearing solution (BioLegend, 427704) while sections on slides were mounted in Mowiol (Sigma, 81381) and coverslipped (VWR, #631-0147) for imaging. Pharmaceutical intervention prior to behaviour testing To reduce dI3 neurons activity, JHU37160 (dihydrochloride, DREADD ligand, Hello Bio, HB6261) was administered subcutaneously at a dosage of 0.5mg/kg. For dI3 ON groups, the equivalent dosage/volume was administered as saline subcutaneously. To modulate cholinergic neurotransmission, mecamylamine (hydrochloride, Cayman Chemical, 826-39-1), was administered intraperitoneally at a dosage of 5mg/kg to effectively reduce Renshaw cell activation, while the equivalent volume of saline was injected for saline condition 39 , 96 . The mecamylamine treatment, was used in combination with either JHU37160 or saline to create 4 treatment conditions, listed as follows: dI3 ON + saline, dI3 ON + mecamylamine, dI3 down + saline and dI3 down + mecamylamine. All mice performed behavioural sessions for the 4 treatment conditions. These sessions were separated by at least 48 hours to allow for complete drug metabolism and to prevent hM4D(Gi) receptor desensitization. Training regimen and behavioural testing Following recovery after electrode implantation surgeries, all mice were given 2 weeks (total of 10 sessions) of training and acclimation to the testing apparatuses and behavioural testing room. The first week consisted of strictly treadmill training. The second week combined both treadmill training and acclimation of the other testing apparatuses. For acclimation to the other apparatus, mice were placed on a horizontal ladder with all ladder rungs in place and evenly spaced and then on the balance beam, without vibration. Following training and acclimation (∼4-5 weeks after intraspinal injections), 9-12 weeks old mice were tested on three different behavioural tasks. For treadmill trials, mice were placed on a horizontal treadmill set to 0.04m/s and walked for approximately 5 minutes. For half of the total treadmill sessions, electrical perturbations were triggered at the onset of the swing phase via electrical stimulation of the saphenous nerve. Treadmill trials were then repeated for the remaining half of behavioural sessions with mechanical perturbation with the use of a thin metal rod to physically perturb the swing of the hindlimb during walking. Electrical stimulation of the saphenous nerve was performed using live EMG recordings of the TA muscle from a custom closed-loop Axoscope protocol (Molecular Devices), wherein threshold detection of bursting activity from the TA muscle triggered an electrical pulse sent to the saphenous nerve cuff implanted in the left hindlimb (0.2ms duration, 100Hz, ranging from 0.1-0.3mA). Stimulus intensity of the pulse to the saphenous nerve was determined individually for each mouse as 1.2 times the required current to elicit a small response from the TA muscle at rest. Throughout the duration of treadmill trials, muscle activity from TA and LG hindlimb muscles were recorded simultaneously with Axoscope software (Molecular Devices, 10 kHz sampling frequency). For each condition, on each mouse, stumbling corrective reaction was triggered at least twice by saphenous nerve stimulation and twice by mechanical perturbation. The maximum step heights was then averaged per mouse for each condition. Mice were then tested on a horizontal ladder and balance beam tests, both 60cm long. In each testing session, mice performed on average 6 attempts with a successful crossing of the ladder and beam. To increase the difficulty of these tasks, the horizontal ladder was set up with unevenly spaced rungs and the balance beam, 0.9cm wide, was equipped with a coin flat vibrating motor (12000rpm, Blinli Amazon Shop). The horizontal ladder rungs were uniquely rearranged prior to each testing session to prevent a learning effect. All videos were recorded at 100 fps for stumbling corrective reactions, horizontal ladder and beam walking (Basler, ace U acA640-750um area scan camera, Cat.# 106748). Imaging and analysis All images were obtained using a Zeiss LSM800 confocal microscope. Images of the hemisections or of the entire coronal sections were obtained using x10 or x20 air objectives (0.3 and 0.8 NA respectively). High magnification images to visualize and quantify presynaptic boutons were taken using a 63x oil objective (1.4 NA). Tiles were stitched using Zen Blue (ZEN Blue 2.3 software) and analyses were performed using Imaris (Bitplane, version 9.6.1) and ImajeJ (version, 1.54k) softwares. All the anatomical analysis on dI3 neurons were performed systematically from L3 to L6. The quantification of presynaptic boutons was performed manually, following the creation of a colocalization channel for PV/vGlut1. Since, Isl1 is also expressed in sensory but not corticospinal neurons, PV OFF /vGlut1 ON /tdTom ON could be identified as putative cutaneous boutons. A colocalization channel was also created for Calb/tdTom or vGlut2/tdTom to analyse putative Renshaw cell and dI3 boutons onto motor neurons respectively. In each case, the colocalization channel was used to facilitate the visualisation of boutons of interest and the expression of individual markers was subsequently confirmed on every bouton quantified. Cell mappings following ΔG-RV injections were manually performed on every other section, in order to minimize the risk of counting the same cell twice in two consecutive sections. Other analyses were carried out on one every 4 slices from L3 to L6 to assess the rostro-caudal variation of the quantified parameters. Distribution maps were plotted setting the central canal as (0,0) in the (x,y) Cartesian system and using the ‘Spots’ function of Imaris. The y-axis was set to the dorso-ventral axis. Positive values were assigned for dorsal neurons in the y-axis and ipsilateral neurons in the x-axis. Coordinates were normalized through the cervical and lumbar parts separately using white mater borders and fixing the width and the height of the transverse hemisections 47 . The normalization of coordinates was performed independently for each quadrant using the following reference points: the x dimension was normalized to the outer edge of the white matter at the level of the central canal, while the y dimension was normalized for each quadrant using the outermost points of the white matter. The resulting cylindrical reconstruction of the spinal cord was then scaled to an idealized spinal cord size for illustrational purposes. All coordinate scaling and mapping were performed using a custom script in R adapted to read .csv files generated by Imaris (R Foundation for Statistical Computing, Vienna, Austria, 2005, http://www.r-project.org , version 3.6.2). When presynaptic boutons on dI3 neurons were imaged, the hemislices were first acquired with a x10 or x20 air objective, and the position of dI3 neurons taken at high magnification were pinpointed on the hemislice to recover their localisation following quantification performed on high magnification images (x63 objective). To observe homonymous/heteronymous proprioceptive boutons onto premotor dI3 neurons following RV injections, the ipsilateral hemislices were first imaged with a x20 objective after staining Isl1, GFP and mCherry and subsequently imaged at high magnification with a x63 oil objective after the additional reaction of PV and vGlut1 to quantify the boutons on previously pinpointed premotor Isl1 positive neurons. The first step of imaging allowed to identify premotor dI3 and reimaged them during the second round of imaging based on their position and morphology, with occasionally the Isl1 staining that was still visible. All video recordings were analysed with DeepLabCut Python software 97 (Version: 2.3.10) to label and track the joints of the hindlimb, and to extract automated hindlimb coordinate data to assess stepping and gait kinematics. Pose estimation data from DeepLabCut was then analysed with the ALMA toolbox Python software 98 (Automated Limb Motion Analysis; Version: 1.1.0) to score the number of paw slips for each attempt across the horizontal ladder and balance beam, as well as to extract various kinematic parameters of stepping on the treadmill. Statistical analysis Statistical analysis were performed using GraphPad Prism (v7), R and RStudio (v1.4.1717). The difference between groups were considered significant for p < 0.05. The statistical tests used are define in the figure legend and the level of significance are reported as follow: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns. indicates no significance. The data produced using electrophysiological or anatomical methods have an intrinsically nested structure, in which observations are taken from the cells, in a specific animal including sometimes an intermediate level, the root stimulated during in vitro recordings. Therefore, to analyse the data collected, we first identified the origin of the variance in our datasets. The interclass correlation coefficient (ICC) was calculated using a one-way random effect ICC model (1,1) with the ICCbare function of the ICC R package. When ICC≤0.5 in all the groups to be compared, we treated the data as independent and applied all the following tests to the lowest level of observation, i.e. the cell. When ICC≥0.5 in at least one of the group, we performed a hierarchical bootstrap. We first resampled with replacement the animals (first level) and then within each animal resampled the cells with replacement (second level) 47 , 99 . Since the ICC between the root stimulated (L4 and L5) and the cell levels was very low in all our datasets, this hierarchical level was not taken into account in our analysis. After ICC calculation, the normality of the distributions was tested using a Shapiro-Wilk tests. The groups compared from electrophysiological datasets were considered as paired for all the parameters as the responses were measured for excitation and inhibition in the same recorded cell following the stimulation of the same pool of axons (ventral or dorsal L4 and L5 roots). For one parameter, when a measurement was possible solely for the inhibitory or the excitatory response following a root stimulation, the cell was excluded from the analysis and the plots shown as there was no pairing possible. For anatomical datasets, the groups were independent. Accordingly, the bootstrap replicas were computed with paired and non-paired resampling and the Hedges’s G coefficient with or without pairing using the R function cohens.d from the “effsize” package. Where mean differences are reported, it corresponds to the distribution of 5 000-10 000 bootstrap replicas, the mean and the 95% CI of the distribution. For behavioural data, despite the hierarchical structure of the data (perturbation or trial/animal), p values were calculated using as statistical unit the animal to benefit from the longitudinal nature of the study. Shapiro-Wilk tests were performed to assess for normality of distribution and repeated measure two way ANOVA followed by Tukey’s multiple comparisons were computed on the summary data using GraphPad Prism. Bootstrapped resampling was here restricted to the computation of Hedge’s G coefficient as the resampling with replacement drastically modify the normality of the distribution given the small number of statistical units. The Hedge’s G reported correspond to the median of 5000 bootstrap replicas that were performed with a resampling with replacement. All Hedge’s G coefficient are reported as absolute values. When bootstrapping was used for computing p values, the reported p values correspond to the median of the distribution. Declaration of interests R.M.B. is a co-founder and director of Sania Therapeutics Inc. Acknowledgements We thank Susan Morton for providing the Isl1 antibody and Julie Tremblay for her assistance with animal care. This work was supported by a Royal Society Newton International Fellowship (NIF\R1\192316) to M.G.O.; a Sir Henry Wellcome Postdoctoral Fellowship (221610/Z/20/Z) to F.N.; Canadian Institute of Health Research Project Grants (PJT 180556) to T.V.B. and (PJT 162357) to T.A.; a Wellcome Trust Discovery Award (227433/Z/23/Z) to R.M.B. and M.B.; a Biotechnology and Biological Sciences Research Council Research Grant (BB/S005943/1) to M.B. and a Wellcome Trust Early Career Award (225674/Z/22/Z) to R.R. Funder Information Declared Wellcome Trust, https://ror.org/029chgv08 , 221610/Z/20/Z , 227433/Z/23/Z , 225674/Z/22/Z Royal Society , NIF\R1\192316 Canadian Institutes of Health Research, https://ror.org/01gavpb45 , PJT 180556 , PJT 162357 Biotechnology and Biological Sciences Research Council , BB/S005943/1 References 1. ↵ James , S.L. , Lucchesi , L.R. , Bisignano , C. , Castle , C.D. , Dingels , Z. V , Fox , J.T. , Hamilton , E.B. , Henry , N.J. , Krohn , K.J. , Liu , Z. , et al. ( 2020 ). 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Share Evidence of spinal cord comparator modules for rapid corrections of movements Gorkem M. Ozyurt , Sarah Chiasson , Alex M Laliberte , Filipe Nascimento , Emam Khan , William P. Mayer , Gardave S. Bhumbra , Turgay Akay , Tuan V. Bui , Marco Beato , Robert M. Brownstone , Rémi Ronzano bioRxiv 2025.08.27.672590; doi: https://doi.org/10.1101/2025.08.27.672590 Share This Article: Copy Citation Tools Evidence of spinal cord comparator modules for rapid corrections of movements Gorkem M. Ozyurt , Sarah Chiasson , Alex M Laliberte , Filipe Nascimento , Emam Khan , William P. Mayer , Gardave S. Bhumbra , Turgay Akay , Tuan V. Bui , Marco Beato , Robert M. 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