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. 2017 Dec 20;96(6):1419-1431.e5.
doi: 10.1016/j.neuron.2017.11.011. Epub 2017 Dec 7.

RORβ Spinal Interneurons Gate Sensory Transmission During Locomotion to Secure a Fluid Walking Gait

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Free PMC article

RORβ Spinal Interneurons Gate Sensory Transmission During Locomotion to Secure a Fluid Walking Gait

Stephanie C Koch et al. Neuron. .
Free PMC article

Abstract

Animals depend on sensory feedback from mechanosensory afferents for the dynamic control of movement. This sensory feedback needs to be selectively modulated in a task- and context-dependent manner. Here, we show that inhibitory interneurons (INs) expressing the RORβ orphan nuclear receptor gate sensory feedback to the spinal motor system during walking and are required for the production of a fluid locomotor rhythm. Genetic manipulations that abrogate inhibitory RORβ IN function result in an ataxic gait characterized by exaggerated flexion movements and marked alterations to the step cycle. Inactivation of RORβ in inhibitory neurons leads to reduced presynaptic inhibition and changes to sensory-evoked reflexes, arguing that the RORβ inhibitory INs function to suppress the sensory transmission pathways that activate flexor motor reflexes and interfere with the ongoing locomotor program. VIDEO ABSTRACT.

Keywords: RORβ; locomotion; motor control; presynaptic inhibition; proprioception; sensory feedback; spinal interneurons.

Figures

Figure 1
Figure 1. Characterization of RORβ IN subpopulations in the spinal cord
(A-C) Sections through the lumbar spinal cord of RORβCre; Thy1∷LSL-YFP mice showing the location of the RORβ INs at P0 (A) and P21 (B-C). At P21, the RORβ INs (green) have formed are localized to dorsal laminae V-VI and lamina III (B), with the dorsal subpopulation of RORβ INs located ventral to PKCY+ INs in lamina IIi (red) and CGRP+ afferents in lamina I (blue) (C). (D) Section through P42 RORβCre; Thy1∷LSL-YFP spinal cord counter-stained with a RORβ in situ hybridization probe (red). (E-F) High magnification images of highlighted sections in (E, lamina III), and (F, lamina V/VI) showing the YFP reporter (green) is largely co-expressed with RORβ (arrowheads). (G) Quantification of the overlap between YFP-positive neurons and RORβ mRNA positive neurons (n=4 cords). (H) Schematic of RORβ IN subpopulations. (I-K) Transverse section through a P42 RORβCre; Thy1∷LSL-YFP spinal cord showing YFP (green) is co-expressed with GAD1 and GlyT2 (red). (J-K) High magnification images from (I) showing coexpression of YFP and GAD1/GlyT2 mRNA (arrowheads) in lamina III (J) and laminae V-VI (K). (L) Quantification of excitatory and inhibitory marker expression (see also Figure S1). RORβCre; R26LSL-HTB; GAD67∷GFP and RORβCre; R26LSL-HTB; GlyT2∷GFP mice were used for the GAD1+/RORβ+ and GlyT2+/RORβ+cell counts, respectively. (M) Transverse section through a P42 RORβCre; Thy1∷LSL-YFP spinal cord showing GAD2 mRNA (red) expression in lamina V/VI RORp INs (green). Inset shows high magnification image of the overlap between RORβ-YFP and GAD2 mRNA in lamina V (arrowheads). (N) Quantification of YFP+/GAD2+ INs in lamina III and laminae V/VI. Scale Bar: 100 pm (A-D, I), 50 μm (E-F, J-K, M (insert)). See also Figure S1.
Figure 2
Figure 2. Inactivating RORβ in spinal inhibitory interneurons causes hyperflexion of the hindlimbs during stepping
(A-F) Still images from high-speed kinematic videos showing the position of the left hindlimb at mid-swing phase with corresponding schematics of genetic expression patterns. The mice shown have the following genotypes: (A) control, (B) RORβ−/−; (C) Nestin∷Cre; RORβfl/−, (D) Emx1Cre; RORβfl/fl, (E) RORαCre; RORβfl/fl, (F) Pax2∷Cre; RORβfl/fl. The schematics associated with each panel show Cre expression, except for (B), which depicts the expression pattern of RORβ. Control mice show a normal walking gait (A, see Movie S1). Note the duck-gait phenotype when RORβ is inactivated in inhibitory Pax2+ INs (F, see Movie S2), but not in RORαCre-derived INs or in cortical Emx1Cre-derived neurons (D and E). See also Figure S2.
Figure 3
Figure 3. Inactivating spinal RORβ interneurons recapitulates the RORβ mutant motor phenotype
(A) Quantification of maximum and minimum hip joint angles during stepping for Pax2-RORβ mutant, RORβ IN-ablated (RORβCre; hCdx2∷FlpO; Tauds-DTR) and RORβ IN-silenced (RORβCre; R26LSL-TeNT) mice and their respective littermate controls (Pax2-RORβ, control: n=8; mutant: n=6; RORβCre; hCdx2∷FlpO; Tauds-DTR, control: n=5; mutant: n=5; RORβCre; R26LSL-TeNT: control: n=5 ; mutant: n=5). In all instances, the minimum hip angle of was significantly decreased compared to their littermate controls (*p < 0.05, **p < 0.01, ***p < 0.001) (B) Quantification of maximum and minimum ankle joint angles during stepping for Pax2-RORβ, RORβCre; hCdx2∷FlpO; Tauds-DTR and RORβCre; R26LSL-TeNT mice and their respective littermate controls. The minimum ankle angle of the Pax2-RORβ mutants was significantly decreased compared to controls (***p < 0.001). (C) Representative stick figure diagrams showing two complete hindlimb step cycles (swing and stance) for (top to bottom): control (grey), Pax2-RORβ (red), RORβCre; hCdx2∷FlpO; Tauds-DTR (green), and RORβCre; R26LST-TeNT (blue) mice. Asterisks indicate the flexion phase. (D) Still image from a high-speed kinematic video showing the position of the left hindlimb at midswing phase after synaptic silencing of RORβ INs in a RORβCre; R26LST-TeNT mouse. (E) Still image from a high-speed kinematic video showing the position of the left hindlimb at midswing phase two weeks after ablation of caudal RORβ INs in a RORβCre; hCdx2∷FlpO; Tauds-DTR mouse. See also Figure S3.
Figure 4
Figure 4. Inactivation of RORβ in inhibitory interneurons disrupts motor neuron excitability and presynaptic inhibition
(A) Recorded VRP traces of dorsal root-evoked VRPs for P8 control and Pax2-RORβ mutant spinal cords. (Inset) Schematic of recording set up. (B-D) Quantification of the area under the curve of the VRP (B). Panels C and C show latency to the polysynaptic VRP (C), and threshold for VRP recruitment (D) (control: n=6 cords; Pax2-RORβ mutant: n=5 cords). Note the lower threshold for VRP recruitment in the Pax2-RORβ mutant cord. (E) Recorded traces showing primary afferent fiber depolarization (PAD) in P8 control and Pax2-RORβ mutant cords. (Inset) Schematic of recording set up. (F-G) Quantification of the amplitude of the PAD (F) and threshold to PAD recruitment (G) (control: n=10; Pax2-RORβ mutant: n=9). The Pax2-RORβ mutant cords displayed decreased PAD compared to control cords. (H) Quantification of the number of parvalbumin (PV) and vGluT 1 double positive terminals that are contacted by GAD2+ boutons in the lumbar cord (laminae V-VI) of P14 control and Pax2-RORβ mutant mice (control: n=4; Pax2-RORβ mutant: n=4). *p < 0.05, **p < 0.01. Scale bar as marked. See also Figure S4.
Figure 5
Figure 5. Inhibitory RORβ interneurons presynaptically inhibit myelinated flexor afferents
(A) Recorded traces of primary afferent fiber depolarization (PAD) from isolated spinal cords of RORβCre; R26LSL-Ai32 mice. (Inset) Schematic of recording set up. Optogenetic activation of RORβ INs (light blue trace) evokes PAD, highlighting RORβ IN-induced presynaptic inhibitory control of primary afferents. (B) Quantification of PAD amplitude following optogenetic activation of RORβ INs at 23°C (n=6 cords). (C) Quantification of RORβ optogenetically-induced PAD at baseline, after application of the GABAergic antagonist bicuculline, and after drug washout, showing RORβ IN presynaptic inhibitory PAD is GABAAR mediated (n=7 cords). (D) Quantification of PAD amplitude following optogenetic activation of RORβ INs at 23°C (left), at 33°C (middle) and after normalization to baseline 23°C recording temperature (right) (n=6 cords). The decrease in amplitude following temperature increase suggests inhibition is predominantly onto large myelinated afferents (see text). (E-F) Transverse spinal section of P14 RORβCre; Thy1∷LSL-YFP immunostained with antibodies raised against GAD2 (red) and PV (blue) highlighting RORβ axo-axonic inhibitory synapses onto myelinated afferents (arrows). (G) Quantification of Thy1+/VGAT+ double positive contacts onto CTb+ labeled afferent terminals from the hip flexor (iliopsoas) or hip extensor (biceps femoris) respectively. Counts were performed in the intermediate lumbar cord of P14 RORβCre; Thy1∷LSL-YFP mice (flexor: n=3; extensor: n=3; p < 0.001). RORβ INs preferentially target hip flexor afferents over hip extensors. 2T, 2x threshold for PAD recruitment; DR, dorsal root. Scale Bar: 5 pm (E-F), and as marked in A. See also Figure S5.
Figure 6
Figure 6. Inactivation of TrkB in RORβ neurons recapitulates the RORβ mutant duck gait phenotype
(A) Transverse section through a P42 RORβCre; Thy1∷LSL-YFP spinal cord showing TrkB mRNA expression (red). Inset shows high magnification image of the overlap between RORβ- YFP and TrkB mRNA in lamina V (arrowheads). (B-C) Still images from high-speed kinematic videos showing the position of the left hindlimb at midswing phase in (B) control and (C) after selective deletion of the BDNF receptor TrkB from RORβ INs using RORβCre; TrkBfl/fl mice. (D-E) Quantification of maximum and minimum joint angles in the hip (D), and ankle (E) during locomotion in control and RORβCre; TrkBfl/fl mice, showing hyperflexion of the hindlimb as seen with RORβ mutant mice (n=5 for each genoptype). (F) Schematic of TrkB-expressing RORβ INs, which presynaptically target BDNF-expressing proprioceptors. (G-H’) Transverse spinal sections of control (G), and RORβCre; TrkBm (H) mice immunostained with antibodies to GAD2 (red) and vGluT1 (blue). Note the GAD2+ inhibitory contacts onto large vGluT1 terminals in laminae V-VI (arrowheads). (I) Number of vGluT1+ terminals containing GAD2+ boutons in laminae V-VI of P42 control and RORβCre; TrkBfl/fl spinal cords (control: n=3; mutant: n=3). YFP immunofluorescence (green) was visualized without amplification. *p < 0.05, **p < 0.01, ***p < 0.001. Scale Bar: 5 pm (G-H’).
Figure 7
Figure 7. Blocking peripheral nerve transmission attenuates the RORβ mutant duck gait phenotype
(A-B) Still images from high-speed kinematic videos showing the position of the left hindlimb at midswing phase in a Pax2-RORβ mutant mouse at (A) baseline and (B) 10 minutes after perisciatic anesthetic injection. (C) Quantification of the maximum and minimum hip joint angles in control and Pax2-RORβ mutants at baseline and 10 minutes after applying either a cutaneous or perisciatic blockade. Pax2-RORβ mutants show a normalization of hip joint movements after perisciatic anesthetic injection (control: n=5; Pax2-RORβ mutant: n=5). (D) Quantification of step cycle duration after sensory blockade in control and Pax2-RORβ mutants at baseline and 10 minutes following perisciatic blockade. Peripheral sensory blockade significantly decreases the duration of the swing phase in Pax2-RORβ mutant mice compared to their baseline measurements. *p < 0.05, **p < 0.01

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