Long-Distance Descending Spinal Neurons Ensure Quadrupedal Locomotor Stability  Ludwig Ruder, Aya Takeoka, Silvia Arber  Neuron  Volume 92, Issue 5, Pages 1063-1078 (December 2016) DOI: 10.1016/j.neuron.2016.10.032 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Neurotransmitter Identity Subdivides Cervico-Lumbar Projection Neurons (A) Experimental strategy to visualize axonal and synaptic patterns of cervical spinal neurons projecting to lumbar spinal segments. Unilateral intraspinal cervical injection of AAV-flex-Tag viruses into mice expressing Cre recombinase from excitatory (vGlut2) or inhibitory (vGAT) neurotransmitter locus (NT::Cre) to assess axon number in white matter at T1 and L1 on transverse sections. (B) Percentage of T1 axons reaching L1 spinal levels (vGlut2, n = 4 mice; vGAT, n = 3 mice), of experiments shown in (A). (C) Axon counts at L1 upon cervical injections into vGlut2Cre and vGATCre mice (vGlut2, n = 4 mice; vGAT, n = 3 mice). (D and E) Representative reconstruction of axon tract distribution at L3 (left) in vGlut2Cre (D; n = 4 mice) and vGATCre (E; n = 3 mice) mice with quantitative assessment of ipsi- and contralateral ratios (right). (F–I) Representative images (F and G), distribution of synaptic density (left; including dorso-ventral and medio-lateral densities), and dorso-ventral and ipsi-contralateral ratios (right) (H and I) of SynTag terminals at L3 upon cervical spinal cord injection into vGlut2Cre (F and H; n = 8 mice) and vGATCre (G and I; n = 6 mice) mice. See also Figure S1. Neuron 2016 92, 1063-1078DOI: (10.1016/j.neuron.2016.10.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Restricted Spinal Distribution of Cervico-Lumbar Projection Neurons (A) Experimental strategy to visualize cell body distribution of cervical-lumbar projection neurons by Rab-FP injection into the lumbar spinal cord. (B) Representative image of cervical section showing Rab-FP marked cervico-lumbar projection neurons (purple) and ChAT (yellow). (C and D) Reconstruction of Rab-FP marked cervico-lumbar projection neurons (C) and quantification of dorsal and ventral populations (D; n = 3 mice), using a boundary of 150 μm dorsal to the central canal position (dotted horizontal line) as a cutoff for analysis (corresponding approximately to the ventral boundary of the dorsal funiculus). All subsequent contour plot reconstructions shown in this figure (E–G and I) use this cutoff for analysis and focus on the ventral population, since neurons in these locations are genetically accessible through progenitor domain origin and have demonstrated locomotor functions. (E) Contour plot for distribution of ventral cervico-lumbar projection neurons marked by Rab-FP, including dorso-ventral and medio-lateral density distributions (n = 3 mice). (F) Experimental strategy (left) and contour plot of reconstructions (right) for ventral cell bodies of AAV-flex-Tomato marked cervico-lumbar projection neurons (n = 6 mice). (G) Contour plots of reconstructions for ventral vGlut2ON and vGATON cervico-lumbar projection neurons, infected by EnvA-coated Rab-FP from the lumbar spinal cord through conditionally expressed TVA in cervical neurons (right, experimental scheme). (H–K) Representative image of Rab-FP infected cervico-lumbar projection neurons in GlyT2::GFP mice (H), contour plot (I), quantification (J), density from central canal (CC), and position analysis in bins <150 μm and >150 μm from CC (K) for reconstructions of ventral reconstructed population of GlyT2ON and GlyT2OFF cervico-lumbar projection neurons (n = 3 mice). (L) Summary diagram depicting division of cervico-lumbar projection neurons into four classes based on neurotransmitter status (vGlut2/vGAT) and projection/connectivity patterns. See also Figure S2. Neuron 2016 92, 1063-1078DOI: (10.1016/j.neuron.2016.10.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Progenitor Domain Origin Subdivides Cervico-Lumbar Projection Neurons (A) Scheme of spinal cord progenitor domains (dI1-dI6, V0-V3, and MN) and transcription factor code of analyzed domains (dI3-Isl1, V0-Dbx1, V1-En1, V2-Shox2, and V3-Sim1). (B) Cre recombinase expression from progenitor domain transcription factor loci (PD::Cre) and individual mouse lines are crossed to Tau-flex-FLPo mice to achieve permanent expression of FLPo recombinase in neuronal descendants of different progenitor domains. Unilateral cervical spinal injection of AAV-FRT-Tag allows analysis of lumbar projections and synaptic terminals in the lumbar spinal cord. (C) Percentage of axons at T1 segmental levels upon cervical AAV-injections as outlined in (B), reaching T6 and L1 spinal levels for different progenitor domain neuron descendants. Note that of five analyzed lines, only axons of V0 and V2-Shox2 neurons reach lumbar levels (L1) at high numbers (Dbx1, n = 3; Shox2, n = 4; En1, n = 5; Isl1, n = 4; Sim1, n = 1 mice). (D) Representative reconstruction of axon tract distribution at L3 (left) and quantification of ipsi- and contralateral ratios (right) in V0-Dbx1 (n = 3) and V2-Shox2 (n = 4) mice. (E–J) Representative images (E and F), distribution of synaptic density (left, including dorso-ventral and medio-lateral densities), ipsi-contralateral ratios (right) (G and H; Dbx1, n = 3; Shox2, n = 4 mice), and percentage of vGlut2ON terminals (I and J) of SynTag terminals at L3 upon cervical spinal cord injection into V0-Dbx1 (E, G, and I) and V2-Shox2 (F, H, and J) mice. (K) Summary diagram of V0-Dbx1 and V2-Shox2 cervical projection neuron arborizations to the lumbar spinal cord. See also Figure S3. Neuron 2016 92, 1063-1078DOI: (10.1016/j.neuron.2016.10.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Ablation of Cervico-Lumbar Projection Neurons Impairs Exploratory Locomotion (A) Scheme of cervico-lumbar projection neuron targeting strategy and time line for behavioral analyses. (B) Representative plot illustrating the definition of a locomotor bout (light blue window) during open-field exploration with a threshold for locomotor onset (>200 ms at >5 cm/s) and termination (<5 cm/s). (C and D) Quantification of postural instability (C) and representative example traces (D) of locomotor bouts for control (n = 7) and PN-DTR (n = 10) mice before (left) and 14 days after (right) DTX injection. (E) Representative locomotor bout traces for control and PN-DTR mice before and 14 days after DTX injection, displayed in different colors and centered according to initiation time point (white dashed line indicates average distance of locomotor bouts). (F) The five highest speed trials for one representative mouse of each control and PN-DTR group before and 14 days after DTX injection are displayed in a speed versus time plot. (G) Quantification of distance, maximal speed, and duration for control and PN-DTR group before and 14 days after DTX injection. (H) Fractional analysis of locomotor bouts with respect to maximal speed achieved during each of the analyzed bouts for one representative mouse of each control and PN-DTR group before and 14 days after DTX injection (0/6 PN-CON and 8/10 PN-DTR mice with p < 0.05 changes in speed histogram distribution before and 14 days after DTX injection). See also Figure S4. Neuron 2016 92, 1063-1078DOI: (10.1016/j.neuron.2016.10.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Cervico-Lumbar Projection Neuron Ablation Impairs Gait at High Speeds (A and B) Representative example and quantification of interlimb coordination for PN-DTR mice (n = 6) at 40 cm/s treadmill speed before (pre) and after (post) cervico-lumbar projection neuron ablation. Each analyzed step is categorized as gray, yellow, and red depending on the calculated phase value (Satoh et al., 2016) and as defined in the Experimental Procedures. Phase values of right hindlimb (RHL) and left and right forelimb (LFL and RFL) are in reference to the left hindlimb (LHL). Compiled circular phase value plots and bar plots of corresponding experiments displaying the percentage of corresponding categories are shown in (B). (C) Reconstructed fore- and hindlimb trajectories and a representative example of calculated fore- and hindlimb oscillation at 40 cm/s before and after cervico-lumbar projection neurons ablation are shown. (D) Intralimb coordination parameters including the degree of linear coupling of joint oscillation and the consistency of endpoint trajectory are not affected by cervico-lumbar projection neuron ablation. See also Figure S5. Neuron 2016 92, 1063-1078DOI: (10.1016/j.neuron.2016.10.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Cervico-Lumbar Projection Neurons Broadcast Information to Other Targets (A) Scheme of experimental strategy to mark synapses of cervico-lumbar projection neurons, and assess collateralization throughout the spinal cord and to supraspinal structures. (B and C) Synaptic terminals of cervico-lumbar projection neurons are observed throughout the spinal cord (B; n = 6 mice), including at thoracic levels (1), at cervical levels where cell bodies reside (2), at cervical levels rostral to cell body residence (3), and within the brainstem, where these neurons mainly terminate within the lateral reticular nucleus (LRN; 4) and parabrachial nucleus (PB; 5). Neuron 2016 92, 1063-1078DOI: (10.1016/j.neuron.2016.10.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Broad Supraspinal Input to Cervico-Lumbar Projection Neurons (A) Two-step viral experimental strategy to map supraspinal synaptic input to cervico-lumbar projection neurons of different neurotransmitter identity in NT::Cre mice (vGATCre and vGlut2Cre). Possible input from dorsal root ganglia sensory neurons was not assessed. (B) Example images of supraspinal regions providing input to excitatory vGlut2ON cervico-lumbar projection neurons. Rab-FPON neurons are shown in cortex, red nucleus (RN; parvicellular division), vestibular nucleus (Ve), pontine reticular formation (PRF), medullary reticular formation (MRF), and medullary reticular formation, ventral part (MdV). (C) Top-down projection of three-dimensional reconstructions of supraspinal neurons connected to vGlut2ON or vGATON cervico-lumbar projection neurons by using rabies transfer with monosynaptic restriction. Color code for different supraspinal populations is indicated (left) and regional definitions are specified in the Experimental Procedures. (D) Quantification of cortical neuron contribution to all supraspinal neurons (left) and the contribution of subcortical neurons to different structures (right; nomenclature as in C). (E) Anterograde mapping of cortical input to GlyT2ON and GlyT2OFF cervico-lumbar projection neurons by cortical AAV-SynTag injections and retrograde Rab-FP labeling from the lumbar spinal cord in GlyT2GFP mice. Experimental scheme (left), example neurons for Neurolucida reconstructions (middle) and quantification (right, n = 2 mice, bilateral injections, 4–5 neurons per mouse and side of each GlyT2ON and GlyT2OFF population) of cortical input to cervico-lumbar projection neurons. (F) Summary diagram of main supraspinal structures providing input to descending cervico-lumbar projection neurons (color code as in C). See also Figure S6. Neuron 2016 92, 1063-1078DOI: (10.1016/j.neuron.2016.10.032) Copyright © 2016 Elsevier Inc. Terms and Conditions