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Skilled reaching relies on a V2a propriospinal internal copy circuit.

Azim E, Jiang J, Alstermark B, Jessell TM - Nature (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

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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Ablation of C3-T1 V2a INs selectively perturbs reachinga,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 IN 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 INs in C3-T1. Error bars indicate s.e.m. c, No GFP+ V2a INs (arrowheads) were found in mid thoracic or lumbar segments prior to DT administration, and normal numbers of V2a INs 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 (gray). Viral injection did not affect success rate, while 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 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 IN 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 vs. 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 vs. post-DT misses, P < 0.0001, DTR pre-DT misses vs. 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 vs. post-DT misses, P < 0.001, DTR pre-DT misses vs. 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 IN ablation (n = 3 DTR, n = 4 control; twoway repeated-measures ANOVA, F1,5 = 0.088, P = 0.78). i, V2a IN 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 s.e.m.
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Figure 9: Ablation of C3-T1 V2a INs selectively perturbs reachinga,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 IN 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 INs in C3-T1. Error bars indicate s.e.m. c, No GFP+ V2a INs (arrowheads) were found in mid thoracic or lumbar segments prior to DT administration, and normal numbers of V2a INs 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 (gray). Viral injection did not affect success rate, while 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 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 IN 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 vs. 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 vs. post-DT misses, P < 0.0001, DTR pre-DT misses vs. 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 vs. post-DT misses, P < 0.001, DTR pre-DT misses vs. 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 IN ablation (n = 3 DTR, n = 4 control; twoway repeated-measures ANOVA, F1,5 = 0.088, P = 0.78). i, V2a IN 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 s.e.m.

Mentions: We targeted cervical V2a INs for elimination by unilateral injection of a viral vector that directed conditional expression of a diphtheria toxin receptor (DTR)-GFP fusion (AAV-FLEX-DTR-GFP) into C3-T1 levels of Chx10::tdT mice (Fig. 4a, Extended Data Fig. 3a)35. Seven days after viral injection >80% of tdT+ V2a INs selectively expressed DTR-GFP, and the LRN contained a dense network of GFP+ axons (Fig. 4b, Extended Data Fig. 3b). Diphtheria toxin (DT; 400 ng) administration 14-21 days after DTR transduction resulted, 7 days later, in a >80% elimination of C3-T1 tdT+ V2a INs and a virtually complete loss of GFP+ axons within the LRN (Fig. 4c, Extended Data Fig. 3b,c).


Skilled reaching relies on a V2a propriospinal internal copy circuit.

Azim E, Jiang J, Alstermark B, Jessell TM - Nature (2014)

Ablation of C3-T1 V2a INs selectively perturbs reachinga,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 IN 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 INs in C3-T1. Error bars indicate s.e.m. c, No GFP+ V2a INs (arrowheads) were found in mid thoracic or lumbar segments prior to DT administration, and normal numbers of V2a INs 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 (gray). Viral injection did not affect success rate, while 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 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 IN 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 vs. 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 vs. post-DT misses, P < 0.0001, DTR pre-DT misses vs. 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 vs. post-DT misses, P < 0.001, DTR pre-DT misses vs. 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 IN ablation (n = 3 DTR, n = 4 control; twoway repeated-measures ANOVA, F1,5 = 0.088, P = 0.78). i, V2a IN 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 s.e.m.
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Figure 9: Ablation of C3-T1 V2a INs selectively perturbs reachinga,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 IN 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 INs in C3-T1. Error bars indicate s.e.m. c, No GFP+ V2a INs (arrowheads) were found in mid thoracic or lumbar segments prior to DT administration, and normal numbers of V2a INs 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 (gray). Viral injection did not affect success rate, while 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 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 IN 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 vs. 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 vs. post-DT misses, P < 0.0001, DTR pre-DT misses vs. 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 vs. post-DT misses, P < 0.001, DTR pre-DT misses vs. 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 IN ablation (n = 3 DTR, n = 4 control; twoway repeated-measures ANOVA, F1,5 = 0.088, P = 0.78). i, V2a IN 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 s.e.m.
Mentions: We targeted cervical V2a INs for elimination by unilateral injection of a viral vector that directed conditional expression of a diphtheria toxin receptor (DTR)-GFP fusion (AAV-FLEX-DTR-GFP) into C3-T1 levels of Chx10::tdT mice (Fig. 4a, Extended Data Fig. 3a)35. Seven days after viral injection >80% of tdT+ V2a INs selectively expressed DTR-GFP, and the LRN contained a dense network of GFP+ axons (Fig. 4b, Extended Data Fig. 3b). Diphtheria toxin (DT; 400 ng) administration 14-21 days after DTR transduction resulted, 7 days later, in a >80% elimination of C3-T1 tdT+ V2a INs and a virtually complete loss of GFP+ axons within the LRN (Fig. 4c, Extended Data Fig. 3b,c).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus