Spinal dI2 interneurons regulate the stability of bipedal stepping

Peripheral and intraspinal feedback is required to shape and update the output of spinal networks that execute motor behavior. We report that lumbar dI2 spinal interneurons of the chick receive synaptic input from afferents and pre-motoneurons. They innervate contralateral premotor networks in the lumbar and brachial spinal cord and their ascending projections innervate the cerebellum. These findings suggest that dI2 neurons function as interneurons in local lumbar circuits and involved in lumbo-brachial coupling and that part of them deliver peripheral and intraspinal feedback to the cerebellum. Silencing of dI2 neurons leads to destabilized stepping in P8 hatchlings with occasional collapses, variable step-profiles and wide-base walking, suggesting that the dI2 neurons may contribute to stabilization of the bipedal gait.


Introduction 50
The spinal cord integrates and relays the somatosensory inputs required for further 51 execution of complex motor behaviors. Interneurons that differentiate at the ventral 52 progenitor domain, V3-V0, are involved in the control of rhythmic motor activity, alternating 53 between left and right limbs, as well as between flexor and extensor muscles (Lai et  neurons are located at the medial lamina VII at the lumbar level (LS3) (C) and the thoracic level (T1) (D). dI2 axons cross the floor plate (yellow arrowheads), turn longitudinally at the ventral funiculus (white arrowheads) and eventually elongate at the lateral funiculus (white arrows). E. A graph indicating the fraction of dI2 neurons expressing TFs during development (based on data from three E5, two E6 and two E14 embryos). F. Cross section of an E17 embryo at the lumbar spinal cord (crural plexus level, LS2). Small-diameter dI2 neurons residing in lamina VII (F') and ventromedial large-diameter dI2 neurons in lamina VIII (F"). G. Density plots of dI2 somata in the sciatic plexus level (cyan, N=374 cells), dI2large (magenta) and dI2small (yellow) INs (N=33 and N=344 cells, respectively, from 2 embryos). See Figure S2 and S3.
percentage of Gad2 and Slc6a5 dI2-expressing cells was found also in mouse (Delile et  To study the supraspinal targets of dI2 neurons, axonal and synaptic reporters were 152 expressed in lumbar dI2 neurons ( Fig. 2A). At E3, dI2 enhancers were co-electroporated with 153 double conditional axonal reporter -membrane tethered cherry, and synaptic reporter -154 SV2-GFP (Fig. S1A). Expression into the lumbar spinal cord was attained by using thin 155 electrodes positioned near the lumbar segments. At E17, the stage in which the internal 156 granule layer is formed in the chick cerebellum, the axons and synapses of dI2 neurons were 157 studied. dI2 axons cross the spinal cord at the floor plate at the segmental level, ascend to 158 the cerebellum, enter through the superior cerebellar peduncle, and cross back to the other 159 side of the cerebellum ipsilaterally to the targeted dI2s (Fig. 2B). Synaptic boutons are 160 noticeable in the granule layer at the ipsilateral and contralateral sides of the anterior 161 cerebellar lobules (Fig. 2C). Synaptic boutons were also present in the central cerebellar 162 nuclei (Fig. S4A). 163 The difference in the soma size between the dorsally and ventrally located dI2 164 neurons prompted us to test which dI2 neurons project to the cerebellum. The dI2 and pre-165 cerebellar neurons were co-labeled by genetic targeting of dI2 at early stages of 166 embryogenesis (E3), coupled with intra-cerebellar injection of cholera toxin subunit B (CTB) 167 or replication-defective HSV-LacZ at E15 ( Fig. 2A). Spinal neurons retrogradely-labelled from 168 the cerebellum consist of the double crossed VSCT and the ipsilaterally projecting DSCT 169 neurons. However, dI2 neurons, double labelled by genetic targeting and back labelling from 170 the cerebellum, are all VSCT neurons, since dI2 are commissural neurons. Only the large-171 diameter neurons were co-labeled, most of them in the ventral aspect of lamina VII (Fig.  172 2D,E,H; S4B,C). Interestingly, many of CTB + or LacZ + neurons were contacted by dI2 axons 173 A. Experimental setup for labeling of cerebellar projecting dI2 neurons. dI2 neurons were genetically targeted at HH18, and pre-cerebellar neurons were labeled using intra-cerebellar injection of CTB or replication defective HSV-LacZ at E15. B. A cross section of E17 brainstem and cerebellum. The dashed polygon in B' is magnified in B. dI2 axons reach the cerebellum, enter into it via the superior cerebellar peduncle and cross the cerebellum midline. Calbindin (Purkinje neurons, magenta (B') or red (B)), synaptotagmin (yellow). C. A cross section of E17 cerebellar cortex. Lumbar-originating dI2 synapses (cyan) in the granular layer of the anterior cerebellar cortex. Calbindin (Purkinje neurons, magenta), synaptotagmin (yellow). D. A cross section of an E15 embryo at the lumbar spinal cord level (sciatic plexus level). Pre-cerebellar neurons were infected and labeled by HSV-LacZ (magenta), and dI2 neurons express GFP (cyan). A large-diameter dI2 neuron co-expressing LacZ and GFP is indicated by an arrow (magnification of the two channels in the insets). E. Density plots of dI2 and pre-cerebellar neurons (density values 10-90%) in the sciatic plexus segments (N=374 and N=289 cells, respectively). F. CTB labeled pre-cerebellar neuron (magenta) is contacted by dI2 axonal terminals (cyan). G. Density plots of dI2 synapses and pre-cerebellar neuron somata (density values 10-90%) in the sciatic plexus segments (N=4735 synapses and N=289 cells, respectively). H,I. Quantification of the overlap in area and volume of the two density plots. The plots are based on data from 3 embryos. See Figure S4.

dI2 neurons receive synaptic input from pre-motoneurons and sensory neurons 186
To assess the synaptic input to dI2 neurons we investigated their synaptic connectivity with 187 known pre-VSCT neurons: dorsal root ganglion (DRG) neurons, inhibitory and excitatory pre-   S1) electroporated at HH18. Pre-MNs were labeled by injection of PRV-cherry into the hindlimbs (D, E) or the forelimb (F) musculature, at E13. The embryos were incubated until pre-MNs infection (39 hours). C. dI2 neurons innervate the contralateral dI2 neurons (N=4735 synapses and N=374 cells, respectively). D. dI2 neurons innervate ipsilateral projecting premotoneurons at the sciatic plexus level (N=4735 synapses and N=936 cells, respectively). E. dI2 neurons innervate contralateral projecting premotoneurons at the sciatic plexus level (N=4735 synapses and N=47 cells, respectively). F. dI2 neurons innervate brachial ipsilateral projecting premotoneurons (N=2215 synapses and N=286 cells, respectively). G. Quantification of the overlap area of different synaptic targets and dI2 synapse density plots, as the percentage of overlap between dI2 synapses and the target. H. Quantification of the overlap in volume of the different synaptic targets and dI2 synapse density plots, as the percentage of overlap between the synaptic target and dI2 synapses. See Figure S7.

Silencing of dI2 neurons impairs stability of bipedal stepping 256
The synaptic input to dI2 and their targets, implicate them as relaying information about 257 motor activity to the contralateral spinal cord and the cerebellum. Thus, we hypothesized 258 that manipulation of their neuronal activity may affect the dynamic profile of stepping. 259 To study the physiological role of dI2 neurons, we silenced their activity using bilateral 260 targeting of the tetanus toxin (TeTX) gene, to lumbar dI2s. EGFP was co-targeted in a 2/1 261 The weight of all chicks was comparable within the range of 144.7±12.1 gr (Supp. 274 Table S1). As a functional measure of foot grip, we tested the ability of the chicks to maintain Analysis of over ground locomotion of the control and TeTX treated chicks revealed 282 no significant differences is swing velocity and striding pattern. An 180 0 out-of-phase pattern 283 was found during stepping in all the manipulated and the control chicks (Fig. S8A, table 1,  284 Supplementary statistics). However, substantial differences were scored in stability 285 parameters: TeTX chicks exhibited whole-body collapses during stepping (Fig. 5B,C, Fig. 6A), 286 wide-base stepping (table 2), and variable limb movements (Fig.5 D,E, Fig. 6B,C, Fig. S8B,C  287 ,tables 3, Supplementary statistics). 288 Whole-body collapses: A collapse was scored as a decline of the knee height below 85% of 289 the average knee height at the stance phase of the step (arrow in Fig. 5C). Collapses were 290 usually followed by over-extensions (arrow head in Fig. 5C). We measured the number of 291 collapses in 50-190 steps. In control chicks, collapses occurred in 0.53±0.92% of the steps. In 292 TeTX manipulated chicks we observed collapses in 19.46±8.3% of the steps, significantly 293 different from the controls (Fig. 6A, Supplementary statistics). The collapses and over-294 extensions were also manifested in the profiles of the knee height trajectory during the 295 swing phase (Fig. 5D). 296 Wide base stepping: Wide-base stance is typical of an unbalanced ataxic gait. The stride 297 width was measured between the two feet during the double stance phase of stepping. The 298 mean stride in TeTX-manipulated chicks 1,2,4, and 5 was significantly wider than the control 299 chicks, while the width in TeXT3 was similar to the controls (   Fig. 5D and Fig. 5E, respectively. These data demonstrate that the range of changes in 308 TeTX-manipulated chicks was higher comparing to control chicks. 309 Further analyses revealed that overall the control group shows lower Knee-height 310 and TMP angle ranges than the TeXT-treated group, even though there are differences 311 within groups (Fig. S8B,C). The average knee height range of the combined control chicks 312 (1.981±0.33) is significantly lower than the range of the combined TeXT treated chicks 313 the TeXT-treated chicks (77±22.149). (Fig. 6C, Supplementary statistics). 317 Since the increased range of changes could be due to the effects of the substantial 318 increase in body collapses during stepping (Fig. 6A, see also    The VSCT is thought to provide peripheral and intrinsic spinal information to the cerebellum 349 in order to shape and update the output of spinal networks that execute motor behavior. 350 The lack of genetic accessibility to VSCT neurons hampers elucidation of their role in 351 locomotion. Using genetic toolbox to dissect the circuitry and manipulate neuronal activity Using the intersection between genetic drivers and spatially restricted delivery of 362 reporters to define lumbar and brachial neurons, we have identified several targets of dI2 363 lumbar neurons. Ipsilateral lumbar dI2 neurons innervate contralateral lumbar dI2 neurons 364 as well as commissural and non-commissural lumbar pre-motoneurons. This connectivity 365 may affect the bilateral spinal output circuitry at the lumbar cord (e.g. (Bras et al., 1988, 366 Jankowska and Hammar, 2013)). Moreover, the ascending axons of lumbar dI2s, give off 367 grey matter collaterals innervating contralateral dI2s and commissural and non-commissural 368 pre-motoneurons throughout the brachial spinal cord (Fig. 6D). Therefore, lumbar dI2 369 neurons may also contribute to the inter-enlargement coupling described between the limb 370 and wing moving segments of the spinal cord (e.g. (Valenzuela et al., 1990, Ruder et al., 371 2016) for forelimb to hindlimb coupling connectivity in mice). 372 We demonstrated that lumbar dI2s receive sensory innervation, pre-motor inhibitory 373 and excitatory innervation, and innervation from contralateral lumbar dI2s (Fig. 6D). Thus, 374 lumbar dI2 neurons can provide the cerebellum and contralateral premotor neurons with 375 proprioceptive information, copies of motor commands delivered from the ipsilateral pre-376 motoneurons, and integrated information from contralateral dI2 neurons (Fig. 6D). 377 The unclear genetic origin of physiologically equivalent lumbar VSCT neurons, 378 prevented better understanding of their role in hindlimb locomotion. Our wiring and 379 neuronal-silencing studies implicated dI2 as a significant contributor to the regularity and 380 stability of locomotion in P8 hatchlings. The kinematic analysis of TeTX treated hatchlings, 381 revealed imbalanced locomotion with occasional collapses, increased stride variability, wide 382 base stepping, and variable limb movements during stepping. 383 The mechanisms accounting for the impaired stepping following dI2 silencing are still 384 unknown. One of the possible mechanisms is that silencing the dI2s perturbs the delivery of 385 peripheral and intrinsic feedback to the cerebellum, leading to unreliable updating of the 386 motor output produced by the locomotor networks, thereby impairing the bipedal stepping. The codes for both 3D reconstruction and the density plots analysis were written in Matlab. 422 The density plots were generated based on cross section images transformed to a standard 423 form. The background was subtracted, and the cells were filtered automatically based on 424 their soma area or using a manual approach. Subsequently, two-dimensional kernel density 425 estimation was obtained using the MATLAB function "kde2d". Finally, unless indicated 426 otherwise, a contour plot was drawn for density values between 20% and 80% of the 427 estimated density range, in six contour lines. Force test 498 The muscle strength was evaluated using the measurement of the angle of the fall from a 499 ladder with a gradually increasing-angle. This test was repeated for each chicken at least 3 500 times, and the average falling angle was calculated. 501

502
Behavioral tests and analysis 503 The embryos were bilaterally electroporated, and were then allowed let to develop and 504 hatch in a properly humidified and heated incubator. Afterwards, within 32 hours post 505 hatching, the hatchling chicks were imprinted on the trainer. The P8 chicks were filmed in 506 slow motion (240 fps) freely walking (side and top views). The following parameters were 507 scored: 1) weight, 2) foot grip power, 3) kinematics parameters during overground 508 locomotion: a) swing velocity, b) swing and stance duration, c) phase of footfalls, d) height of 509 knee and tarsometatarso-phalangeal (TMP) joints, e) angles of the TMP and ankle joints, and 510 f) stride width (distance between feet during the double stance phase). 511 Using a semi-automated Matlab-based tracking software (Hedrick, 2008), several 512 points of interest were encoded. The leg joints as well as the eye and the tail were tracked. 513 The position of these reference points was used for computational analysis using an in-514 house Matlab code for calculating different basic locomotion parameters (e.g. stick 515 diagrams, velocity, joints trajectory, angles, range, and elevation), steps pattern, and degree 516 of similarity (Haimson B et al. in preparation). Dunnett's test (Dunnett, 1955) was used to 517 perform multiple comparisons of group means following One-way ANOVA. Circular statistics 518 was used for analyses of angular data. Circular statistics was used for analyses of angular 519 data, utilizing Oriana (KCS, version 4).  Figure S1).