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Skilled reaching relies on a V2a propriospinal internal copy circuit

Nature volume 508, pages 357–363 (2014)Cite this article

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Abstract

The precision of skilled forelimb movement has long been presumed to rely on rapid feedback corrections triggered by internally directed copies of outgoing motor commands, but the functional relevance of inferred internal copy circuits has remained unclear. One class of spinal interneurons implicated in the control of mammalian forelimb movement, cervical propriospinal neurons (PNs), has the potential to convey an internal copy of premotor signals through dual innervation of forelimb-innervating motor neurons and precerebellar neurons of the lateral reticular nucleus. Here we examine whether the PN internal copy pathway functions in the control of goal-directed reaching. In mice, PNs include a genetically accessible subpopulation of cervical V2a interneurons, and their targeted ablation perturbs reaching while leaving intact other elements of forelimb movement. Moreover, optogenetic activation of the PN internal copy branch recruits a rapid cerebellar feedback loop that modulates forelimb motor neuron activity and severely disrupts reaching kinematics. Our findings implicate V2a PNs as the focus of an internal copy pathway assigned to the rapid updating of motor output during reaching behaviour.

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Figure 1: Identification of mouse PNs.
Figure 2: Excitatory PNs are a V2a interneuron subpopulation.
Figure 3: Reaching kinematics.
Figure 4: Cervical V2a interneuron ablation perturbs reaching.
Figure 5: Photoactivation of PN terminals in the LRN.
Figure 6: PN terminal photostimulation perturbs forelimb movement via a cerebellar–motor loop.

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Acknowledgements

We thank L. Zagoraiou, S. Crone and K. Sharma for the Chx10-cre mouse; T. Buch for the DTR-GFP construct; K. Deisseroth for the AAV-FLEX-hChR2-YFP plasmid; and M. Churchland for providing a low-pass filter. We are grateful to J. Krakauer for discussion of concepts in mammalian motor control and debate about terminology and text, and T. Isa for comments and continuing discussion on primate forelimb movement. R. Axel, M. Churchland, D. Jabaudon, A. Karpova, A. Miri, N. Sawtell and C. Zuker also provided discussion and comments on the manuscript. We are grateful to K. Miao for technical assistance; S. Fageiry for work on retrograde CTB labeling; T. Akay for assistance with the horizontal ladder task; D. Ng for the Cre-expressing plasmid; M. Mendelsohn, N. Zabello and S. Patruni for animal care; E. Danielsson and P. Utsi for engineering support; and B. Han, K. MacArthur, S. Morton and I. Schieren for technical assistance. E.A. is a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation; J.J. was supported by grants to B.A. from Umeå University; B.A. was supported by grants from the Swedish Research Council; T.M.J. was supported by National Institutes of Health grant NS033245, the Harold and Leila Y. Mathers Foundation and Project A.L.S., and is an investigator of the Howard Hughes Medical Institute.

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  1. Departments of Neuroscience and Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Kavli Institute for Brain Science, Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, New York, 10032, New York, USA

    Eiman Azim & Thomas M. Jessell

  2. Department of Integrative Medical Biology, Section of Physiology, Umeå University, Umeå, Sweden

    Juan Jiang & Bror Alstermark

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  1. Eiman Azim
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E.A., B.A. and T.M.J. designed the experiments and analysed data. E.A. performed molecular, anatomical and behavioural experiments. B.A., J.J. and E.A. performed and analysed electrophysiological experiments. E.A., B.A. and T.M.J. prepared the manuscript.

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Extended data figures and tables

Extended Data Figure 1 LRN-projecting V2a interneurons are restricted to cervical segments.

a, AAV-FLEX-hChR2-YFP was injected unilaterally into C6–T1, mid thoracic and mid lumbar spinal cord of adult Chx10-cre mice. YFP+ projections in the LRN could be identified following C3–C4 (see Fig. 2) and C6–T1 V2a interneuron transduction, but were minimal or absent following thoracic or lumbar transduction. b, Dual injection of CTB into the LRN (red) and WGA into triceps brachii muscle (blue) labelled YFP+ V2a interneurons (green) in intermediate levels of cervical cord in Chx10-cre;Rosa-lsl-YFP mice. CTB also labelled YFPoff neurons, potentially corresponding to inhibitory PNs46.

Extended Data Figure 2 Three-dimensional kinematics of mouse reaching.

a, After training, mice exhibited a high degree of variability in success in the multi-reach assay (37.0% ± 10.4% s.d.; n = 13). Error bars indicate s.d. The mean s.d. of reach success from day to day across mice was 10.2% ± 4.1% s.d. (n = 13). b, Individual reach plots of paw distance to pellet, velocity versus time and velocity versus distance to pellet from a representative mouse. Transition from the early reach phase to the late grab phase of the movement is delineated by the box opening (large dashes). Velocity crossings of zero (small dashes) indicate reversals in direction towards or away from the pellet. See Fig. 3d for mean plots from the same mouse.

Extended Data Figure 3 Ablation of C3–T1 V2a interneurons selectively perturbs reaching.

a, AAV-FLEX-DTR-GFP plasmid DNA was transfected into 293T cells. Only when co-transfected with a Cre-expressing plasmid (red; middle panel) did recombination occur, resulting in expression of DTR (red; right panel) and GFP. b, After viral injection into C3–T1 of adult Chx10::tdT mice, 83% (±0.3% s.e.m.; n = 2) of tdT+ V2a interneuron cell bodies co-expressed GFP and DTR. After DT administration, there was an 84% (±9% s.e.m.; n = 2) reduction in the number of tdT+ V2a interneurons in C3–T1. Error bars indicate s.e.m. c, No GFP+ V2a interneurons (arrowheads) were found in mid thoracic or lumbar segments before DT administration, and normal numbers of V2a interneurons remained following DT administration (arrowheads). d, Success in the multi-reach task quantified by day from a representative DTR-transduced mouse (black) and control mouse (grey). Viral injection did not affect success rate, whereas subsequent DT administration reduced success in the DTR-transduced but not control mouse. See Fig. 4d for mean success rates across mice (pre-DT, 41.3% ± 8.3% s.e.m.; post-DT, 20.7% ± 6.7% s.e.m; n = 3 DTR, n = 4 control; two-way repeated-measures analysis of variance (ANOVA), interaction of group × toxin: F1,5 = 6.67, P = 0.049, post-hoc Bonferroni test; DT: P < 0.05). e, Individual reach trajectories and mean kinematics from a representative DTR-transduced mouse reveal perturbation of trajectory, duration and velocity following ablation. There were no successful reaches in the kinematic assay following V2a interneuron ablation (Supplementary Note 4). See Fig. 4e for individual reach plots from the same mouse. f, Individual and mean reach kinematics from a representative control mouse show no effects of DT administration. Shaded regions indicate s.d. g, In DTR-transduced mice relative to control mice, mean paw velocity decreased (n = 3 DTR, n = 4 control; two-way repeated-measures ANOVA, interaction of group × condition, reach phase: F2,10 = 8.315, P = 0.008, post-hoc Tukey test; DTR pre-DT hits versus post-DT misses, P < 0.01; grab phase: F2,10 = 0.063, P = 0.55) and mean duration of paw movement increased (reach phase: F2,10 = 15.37, P = 0.0009, post-hoc Tukey test; DTR pre-DT hits versus post-DT misses, P < 0.0001, DTR pre-DT misses versus post-DT misses, P < 0.01; grab phase: F2,10 = 0.99, P = 0.40) during the reach phase but not the grab phase following ablation. As shown in Fig. 4f, the mean number of direction reversals increased during the reach, but not the grab, phase in DTR-transduced mice, relative to control mice (reach phase: F2,10 = 19.03, P = 0.0004, post-hoc Tukey test; DTR pre-DT hits versus post-DT misses, P < 0.001, DTR pre-DT misses versus post-DT misses, P < 0.001; grab phase: F2,10 = 2.64, P = 0.12). Shapes represent individual mice and black circles indicate means across mice. See Extended Data Table 1. h, Digit abduction (maximum distance between digits 2 and 4) during grasp attempts was unaffected by V2a interneuron ablation (n = 3 DTR, n = 4 control; two-way repeated-measures ANOVA, F1,5 = 0.088, P = 0.78). i, V2a interneuron ablation had no effect on the mean number of mistakes in right forepaw placement during a horizontal ladder locomotion test (n = 3 DTR, n = 3 control; two-way repeated-measures ANOVA, F1,4 = 3.53, P = 0.13). Moreover, ablation had no effect on the types of mistakes made (stagger, slip or miss; n = 3 DTR, n = 3 control; two-way repeated-measures ANOVA, stagger: F1,4 = 2.49, P = 0.19; slip: F1,4 = 0.41, P = 0.56; miss: F1,4 = 5.17, P = 0.09). Error bars indicate mean ± s.e.m.

Extended Data Figure 4 Selective photoactivation of PN input to the LRN.

a, Population recordings in the LRN revealed photostimulation induced synaptic activation of LRN neurons across a range of optical fibre depths in and above the brainstem. Retraction of the optical fibre from the LRN (presumably resulting in a decrease in light exposure) resulted in a reduced amplitude of the LRN extracellular field potential (arrow), and, consequently in an increase in LRN neuronal firing latency. Schematic depicts coronal section of caudal brainstem and optical fibre depths. IO, inferior olive; MLF, medial longitudinal fasciculus; Pyr, pyramidal tract; Sp5c, spinal trigeminal nucleus, caudal part. b, Extracellular recording of LRN neurons antidromically activated from cerebellum (CB; 20 μA; purple arrows), with the optical fibre just dorsal to the LRN, revealed activation (blue arrows) and spike collision (red arrowheads) across a range of laser intensities (also see Fig. 5a). Increasing the light intensity caused more intense synaptic firing and a slight shortening of the latency from light onset. c, Extracellular recording of LRN neurons in control mice revealed no activation and no collision of the electrically induced antidromic spike from the cerebellum (purple arrows) during photostimulation (n = 0 out of 14 neurons).

Extended Data Figure 5 Photoactivation of PN terminals in the LRN does not elicit antidromic spikes.

a, PNs in C6 were identified antidromically by electrical stimulation from the LRN (40 μA; arrows) and C7 ventral horn (40 μA; not shown) and by spike collision (not shown). b, Cervical photostimulation of the same PN cell bodies activated 69.2% of PNs (n = 9 out of 13; green arrows indicate single spikes), as identified by collision of the LRN antidromic spike (bottom red traces, red arrowheads; compare with antidromic spike in a; two lower black traces exhibit failed collision). c, In the same PNs, photostimulation of PN terminals in the LRN did not trigger antidromic spikes that invaded the cell body (0 out of 31 PNs; 0 out of 3 in control mice), whereas electrical stimulation in the LRN always produced antidromic spikes (lower traces, arrow; compare with antidromic spike in a). Also see Fig. 5b–d and Supplementary Discussion.

Extended Data Figure 6 Selective photostimulation of PN terminals in the LRN perturbs reaching.

a, Mean reach kinematics from a representative mouse with perturbed reach trajectory and large fluctuations in velocity and acceleration during PN terminal photostimulation. See Fig. 6c for individual reach plots from the same mouse. As shown in Fig. 6b, photostimulation reduced success in the multi-reach task in ChR2 versus control mice (no light, 35.7% ± 6.5% s.e.m.; light, 18.3% ± 3.8% s.e.m.; n = 5 ChR2, n = 4 control; two-way repeated-measures ANOVA, interaction of group × light: F1,7 = 8.65, P = 0.02; post-hoc Bonferroni test, ChR2: P < 0.001). There were no successful reaches in the kinematic assay during PN terminal photostimulation (Supplementary Note 4). b, Individual and mean reach kinematics from a representative control mouse show no effects of LRN photostimulation. Shaded regions indicate s.d. c, As shown in Fig. 6d, the mean number of direction reversals during the reach phase increased during photostimulation in ChR2 mice, relative to control mice (n = 5 ChR2, n = 4 control; two-way ANOVA, interaction of group × condition, reach phase: F2,19 = 5.24, P = 0.02; post-hoc Tukey test, ChR2 no light hits versus light misses, P < 0.01, ChR2 no light misses versus light misses, P < 0.01; grab phase: F2,19 = 2.70, P = 0.09). In addition, photostimulation resulted in severe kinematic perturbation during the entire movement (reach and grab phases) in ChR2 mice relative to control mice, including increases in: the mean amount of time spent moving away from the pellet (F2,19 = 4.07, P = 0.03; post-hoc Tukey test, ChR2 no light hits versus light misses, P < 0.01, ChR2 no light misses versus light misses, P < 0.01); the mean minimum distance from the paw to the pellet (F2,19 = 6.37, P = 0.008; post-hoc Tukey test, ChR2 no light hits versus light misses, P < 0.0001, ChR2 no light misses versus light misses, P < 0.001); the mean peak velocity away from the pellet (F2,19 = 9.08, P = 0.002; post-hoc Tukey test, ChR2 no light hits versus light misses, P < 0.001, ChR2 no light misses versus light misses, P < 0.001); the mean s.d. of the velocity (F2,18 = 25.02, P < 0.0001; post-hoc Tukey test, ChR2 no light hits versus light misses, P < 0.0001, ChR2 no light misses versus light misses, P < 0.0001); the mean peak acceleration and deceleration (acceleration: F2,19 = 10.08, P = 0.001; post-hoc Tukey test, ChR2 no light hits versus light misses, P < 0.001, ChR2 no light misses versus light misses, P < 0.0001; deceleration: F2,19 = 21.53, P < 0.0001; post-hoc Tukey test, ChR2 no light hits versus light misses, P < 0.0001, ChR2 no light misses versus light misses, P < 0.0001); and the mean s.d. of the acceleration (F2,18 = 29.21, P < 0.0001; post-hoc Tukey test, ChR2 no light hits versus light misses, P < 0.0001, ChR2 no light misses versus light misses, P < 0.0001). Shapes represent individual mice. Black circles indicate means across mice. See Extended Data Table 2. d, Digit abduction (maximum distance between digits 2 and 4) during grasp attempts was unaffected by photostimulation (n = 5 ChR2, n = 4 control; two-way repeated-measures ANOVA, F1,7 = 3.71, P = 0.10). Error bars indicate mean ± s.e.m.

Extended Data Figure 7 Variance of LRN neuronal spiking and motor neuron EPSPs during PN terminal photostimulation, and evaluation of electrically induced antidromic action potentials in PNs.

a, LRN neurons exhibited low jitter spiking (mean variance 0.009 ± 0.006 ms2 s.d.; n = 9), consistent with monosynaptic input, during photostimulation of PN terminals in the LRN. This contrasts sharply with large motor neuron (MN) EPSP jitter during PN terminal photostimulation (mean variance 0.28 ± 0.35 ms2 s.d.; n = 11), consistent with recruitment of a polysynaptic pathway. An ∼30-fold increase in the variance for motor neuron EPSPs as compared to LRN spiking can be seen. Variance was calculated with respect to the shortest latency response among ten consecutive sweeps. See Fig. 6e for mean motor neuron EPSP onset latency from all ten sweeps. b, Antidromic spiking of PN somata evoked by electrical stimulation of their ascending (LRN, light blue) and descending (C7, dark blue) axonal branches occurred with a mean latency of ∼1 ms in each case, adding to a total conduction time of about 2 ms (black). Subtracting approximate axonal activation and two soma invasion times, which are likely each on the order of 0.4 ms19, provides an estimate for the conduction time of an antidromic action potential across both branches of the PN in the ∼1–1.2 ms range. See Supplementary Discussion.

Extended Data Figure 8 Photostimulation of PN input to the LRN before and after cerebellar lesion.

a, Post-physiology histology confirmed intact inferior cerebellar peduncles (ICP) in control mice and bilateral lesion of inferior cerebellar peduncles (red arrowheads; n = 4) and complete removal of cerebellar cortex and deep cerebellar nuclei (n = 1) in experimental mice. b, Cerebellar lesions resulted in no change in LRN field potential size during PN terminal photostimulation (n = 2; two-tailed paired t-test). c, In non-lesioned mice, C7 field potential recordings revealed that photostimulation of PN terminals in the LRN (black traces) or PN somata in C4 (grey traces) elicited responses restricted to ventral regions of the grey matter, near motor neurons and their dendrites. Field onsets (arrowheads) were consistently later following LRN versus C4 photostimulation (also see Fig. 6e). Schematic depicts axial section of C7 and recording electrode depths. Roman numerals indicate Rexed laminae.

Extended Data Table 1 Pre- and post-V2a interneuron ablation kinematics
Full size table
Extended Data Table 2 Kinematics with and without PN terminal photostimulation
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Supplementary information

Supplementary Information

This file contains Supplementary Notes 1-7, a Supplementary Discussion and additional references. (PDF 198 kb)

Kinematics of a successful reach

High-resolution, high-speed capture of reaching movements from two cameras placed ~80° apart for tracking of infrared-reflective markers attached to the back of the right paw. Plots of three-dimensional (3D) paw trajectory and velocity versus distance to pellet for a successful reach (green) are shown. Pellet location marked with asterisk. Transition from the early reach phase to the late grab phase of the movement is delineated by the box opening (vertical dashes). Velocity crossings of zero (horizontal dashes) indicate reversals in direction toward or away from the pellet. The video is slowed to approximately 6% real time. (MOV 7873 kb)

Kinematics of an unsuccessful reach

Plots of 3D paw trajectory and velocity versus distance to pellet for an unsuccessful reach (brown) by the same mouse shown in Supplementary Video 1. The video is slowed to approximately 6% real time. (MOV 8784 kb)

Kinematics following ablation of C3–T1 V2a interneurons

Plots of 3D paw trajectory and velocity versus distance to pellet for an unsuccessful reach following cervical V2a interneuron ablation (red) by the same mouse shown in Supplementary Videos 1 and 2. Note the higher frequency of paw direction reversals, the increase in reach duration and the reduction in paw velocity during the reach phase (before the box opening). Normal digit abduction occurs as the paw approaches the pellet. The video is slowed to approximately 6% real time. (MOV 36195 kb)

Kinematics with no photostimulation

Plots of 3D paw trajectory and velocity versus distance to pellet for a successful reach (green) following injection of AAV-FLEX-hChR2-YFP and implantation of the fiberoptic ferrule, but with no photostimulation. The video is slowed to approximately 6% real time. (MOV 8868 kb)

Kinematics during PN terminal photostimulation

Plots of 3D paw trajectory and velocity versus distance to pellet for an unsuccessful reach (blue) during photostimulation of PN terminals in the LRN (473 nm, ~12 mW, 20 Hz, 15 ms pulse width) in the same mouse shown in Supplementary Video 4. Note the large increase in the number of direction reversals during the reach phase, the severe effects on trajectory and the large swings in velocity toward and away from the pellet. Normal digit abduction occurs as the paw approaches the pellet. The video is slowed to approximately 6% real time. (MOV 19863 kb)

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Azim, E., Jiang, J., Alstermark, B. et al. Skilled reaching relies on a V2a propriospinal internal copy circuit. Nature 508, 357–363 (2014). https://doi.org/10.1038/nature13021

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