The rich behavioral repertoire of animals is encoded in the CNS as a set of motorneuron activation patterns, also called 'motor synergies'. However, the neurons that orchestrate these motor programs as well as their cellular properties and connectivity are poorly understood. Here we identify a population of molecularly defined motor synergy encoder (MSE) neurons in the mouse spinal cord that may represent a central node in neural pathways for voluntary and reflexive movement. This population receives direct inputs from the motor cortex and sensory pathways and, in turn, has monosynaptic outputs to spinal motorneurons. Optical stimulation of MSE neurons drove reliable patterns of activity in multiple motor groups, and we found that the evoked motor patterns varied on the basis of the rostrocaudal location of the stimulated MSE. We speculate that these neurons comprise a cellular network for encoding coordinated motor output programs.
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Tresch, M.C., Saltiel, P., d'Avella, A. & Bizzi, E. Coordination and localization in spinal motor systems. Brain Res. Rev. 40, 66–79 (2002).
Bizzi, E., Cheung, V.C., d'Avella, A., Saltiel, P. & Tresch, M. Combining modules for movement. Brain Res. Brain Res. Rev. 57, 125–133 (2008).
Duysens, J., De Groote, F. & Jonkers, I. The flexion synergy, mother of all synergies and father of new models of gait. Front. Comput. Neurosci. 7, 14 (2013).
Sherrington, C.S. The Integrative Action of the Nervous System (C. Scribner's Sons, 1906).
Lundberg, A. Supraspinal control of transmission in reflex paths to motoneurones and primary afferents. Prog. Brain Res. 12, 197–221 (1964).
Leyton, S. & Sherrington, C.S. Observations on the excitable cortex of the chimpanzee, orang-utan and gorilla. Q. J. Exp. Physiol. 11, 135–222 (1917).
Graziano, M.S., Taylor, C.S. & Moore, T. Complex movements evoked by microstimulation of precentral cortex. Neuron 34, 841–851 (2002).
McHanwell, S. & Biscoe, T.J. The localization of motoneurons supplying the hindlimb muscles of the mouse. Phil. Trans. R. Soc. Lond. B 293, 477–508 (1981).
Nicolopoulos-Stournaras, S. & Iles, J.F. Motor neuron columns in the lumbar spinal cord of the rat. J. Comp. Neurol. 217, 75–85 (1983).
de Leon, R., Hodgson, J.A., Roy, R.R. & Edgerton, V.R. Extensor- and flexor-like modulation within motor pools of the rat hindlimb during treadmill locomotion and swimming. Brain Res. 654, 241–250 (1994).
Wickersham, I.R., Finke, S., Conzelmann, K.K. & Callaway, E.M. Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat. Methods 4, 47–49 (2007).
Stepien, A.E., Tripodi, M. & Arber, S. Monosynaptic rabies virus reveals premotor network organization and synaptic specificity of cholinergic partition cells. Neuron 68, 456–472 (2010).
Coulon, P., Bras, H. & Vinay, L. Characterization of last-order premotor interneurons by transneuronal tracing with rabies virus in the neonatal mouse spinal cord. J. Comp. Neurol. 519, 3470–3487 (2011).
Ruigrok, T.J., Pijpers, A., Goedknegt-Sabel, E. & Coulon, P. Multiple cerebellar zones are involved in the control of individual muscles: a retrograde transneuronal tracing study with rabies virus in the rat. Eur. J. Neurosci. 28, 181–200 (2008).
Scheibel, M.E. & Scheibel, A.B. Terminal axonal patterns in cat spinal cord. II. The dorsal horn. Brain Res. 9, 32–58 (1968).
Sherrington, C.S. & Laslett, E.E. Observations on some spinal reflexes and the interconnection of spinal segments. J. Physiol. (Lond.) 29, 58–96 (1903).
Sengul, G., Puchalski, R.B. & Watson, C. Cytoarchitecture of the spinal cord of the postnatal (P4) mouse. Anat. Rec. 295, 837–845 (2012).
Harrison, P.J., Jankowska, E. & Zytnicki, D. Lamina VIII interneurones interposed in crossed reflex pathways in the cat. J. Physiol. 371, 147–166 (1986).
Hongo, T., Kitazawa, S., Ohki, Y. & Xi, M.C. Functional identification of last-order interneurones of skin reflex pathways in the cat forelimb segments. Brain Res. 505, 167–170 (1989).
Puskar, Z. & Antal, M. Localization of last-order premotor interneurons in the lumbar spinal cord of rats. J. Comp. Neurol. 389, 377–389 (1997).
Asante, C.O. & Martin, J.H. Differential joint-specific corticospinal tract projections within the cervical enlargement. PLoS ONE 8, e74454 (2013).
Hongo, T., Kitazawa, S., Ohki, Y., Sasaki, M. & Xi, M.C. A physiological and morphological study of premotor interneurones in the cutaneous reflex pathways in cats. Brain Res. 505, 163–166 (1989).
Tresch, M.C. & Bizzi, E. Responses to spinal microstimulation in the chronically spinalized rat and their relationship to spinal systems activated by low threshold cutaneous stimulation. Exp. Brain Res. 129, 401–416 (1999).
Osakada, F. et al. New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits. Neuron 71, 617–631 (2011).
Clarke, R.W. & Harris, J. The organization of motor responses to noxious stimuli. Brain Res. Brain Res. Rev. 46, 163–172 (2004).
Pierrot-Deseilligny, E. & Burke, D. The Circuitry of the Human Spinal Cord. 350–352 (Cambridge University Press, 2012).
Schouenborg, J. & Kalliomaki, J. Functional organization of the nociceptive withdrawal reflexes. I. Activation of hindlimb muscles in the rat. Exp. Brain Res. 83, 67–78 (1990).
Gong, S. et al. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425, 917–925 (2003).
Henry, A.M. & Hohmann, J.G. High-resolution gene expression atlases for adult and developing mouse brain and spinal cord. Mammalian Genome 23, 539–549 (2012).
Wildner, H. et al. Genome-wide expression analysis of Ptf1a- and Ascl1-deficient mice reveals new markers for distinct dorsal horn interneuron populations contributing to nociceptive reflex plasticity. J. Neurosci. 33, 7299–7307 (2013).
Britanova, O., Akopov, S., Lukyanov, S., Gruss, P. & Tarabykin, V. Novel transcription factor Satb2 interacts with matrix attachment region DNA elements in a tissue-specific manner and demonstrates cell-type-dependent expression in the developing mouse CNS. Eur. J. Neurosci. 21, 658–668 (2005).
Alaynick, W.A., Jessell, T.M. & Pfaff, S.L. Snapshot: spinal cord development. Cell. 146, 178 (2011).
Harrison, T.C., Ayling, O.G. & Murphy, T.H. Distinct cortical circuit mechanisms for complex forelimb movement and motor map topography. Neuron 74, 397–409 (2012).
Ramanathan, D., Conner, J.M. & Tuszynski, M.H. A form of motor cortical plasticity that correlates with recovery of function after brain injury. Proc. Natl. Acad. Sci. USA 103, 11370–11375 (2006).
Ishizuka, N., Mannen, H., Hongo, T. & Sasaki, S. Trajectory of group Ia afferent fibers stained with horseradish peroxidase in the lumbosacral spinal cord of the cat: three dimensional reconstructions from serial sections. J. Comp. Neurol. 186, 189–211 (1979).
Scheibel, M.E. & Scheibel, A.B. Terminal patterns in cat spinal cord. 3. Primary afferent collaterals. Brain Res. 13, 417–443 (1969).
Gianino, S. et al. Postnatal growth of corticospinal axons in the spinal cord of developing mice. Brain Res. Dev. Brain Res. 112, 189–204 (1999).
Curfs, M.H., Gribnau, A.A. & Dederen, P.J. Selective elimination of transient corticospinal projections in the rat cervical spinal cord gray matter. Brain Res. Dev. Brain Res. 78, 182–190 (1994).
Martin, J.H. Differential spinal projections from the forelimb areas of the rostral and caudal subregions of primary motor cortex in the cat. Exp. Brain Res. 108, 191–205 (1996).
Schouenborg, J., Weng, H.R., Kalliomaki, J. & Holmberg, H. A survey of spinal dorsal horn neurones encoding the spatial organization of withdrawal reflexes in the rat. Exp. Brain Res. 106, 19–27 (1995).
Saltiel, P., Tresch, M.C. & Bizzi, E. Spinal cord modular organization and rhythm generation: an NMDA iontophoretic study in the frog. J. Neurophysiol. 80, 2323–2339 (1998).
Goulding, M. Circuits controlling vertebrate locomotion: moving in a new direction. Nat. Rev. Neurosci. 10, 507–518 (2009).
Bareyre, F.M., Kerschensteiner, M., Misgeld, T. & Sanes, J.R. Transgenic labeling of the corticospinal tract for monitoring axonal responses to spinal cord injury. Nat. Med. 11, 1355–1360 (2005).
Liang, H., Paxinos, G. & Watson, C. The red nucleus and the rubrospinal projection in the mouse. Brain Struct. Funct. 217, 221–232 (2012).
Brown, L.T. Rubrospinal projections in the rat. J. Comp. Neurol. 154, 169–187 (1974).
Antal, M. et al. The termination pattern and postsynaptic targets of rubrospinal fibers in the rat spinal cord: a light and electron microscopic study. J. Comp. Neurol. 325, 22–37 (1992).
Lundberg, A. Multisensory control of spinal reflex pathways. Prog. Brain Res. 50, 11–28 (1979).
Schomburg, E.D. Spinal sensorimotor systems and their supraspinal control. Neurosci. Res. 7, 265–340 (1990).
Barthelemy, D., Leblond, H., Provencher, J. & Rossignol, S. Nonlocomotor and locomotor hindlimb responses evoked by electrical microstimulation of the lumbar cord in spinalized cats. J. Neurophysiol. 96, 3273–3292 (2006).
Mushahwar, V.K., Aoyagi, Y., Stein, R.B. & Prochazka, A. Movements generated by intraspinal microstimulation in the intermediate gray matter of the anesthetized, decerebrate, and spinal cat. Can. J. Physiol. Pharmacol. 82, 702–714 (2004).
Wickersham, I.R., Sullivan, H.A. & Seung, H.S. Production of glycoprotein-deleted rabies viruses for monosynaptic tracing and high-level gene expression in neurons. Nat. Protoc. 5, 595–606 (2010).
Joshi, P.S. et al. Bhlhb5 regulates the postmitotic acquisition of area identities in layers II–V of the developing neocortex. Neuron 60, 258–272 (2008).
Cheng, L. et al. Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates. Nat. Neurosci. 7, 510–517 (2004).
We acknowledge the generosity and advice of M. Goulding, R. Edgerton, A. Engmann, E. Callaway, J. Young, F. Osakada and F. Stam, P. Joshi and S. McConnell for instruction in cortical injections, and Q. Ma (Harvard Medical School, in situ probes) and C. Birchmeier (Max Delbruck Center for Molecular Medicine, Tlx3 antibody) for providing reagents. M. Sternfeld, M. Hayashi, K. Lettieri and Y. Bogoch provided support and advice. We thank R. Levine for his helpful reading of the manuscript. A.J.L. was supported by George E. Hewitt Foundation for Medical Research and Christopher and Dana Reeve Foundation. C.A.H. was supported by a US National Research Service Award fellowship from US National Institutes of Health NINDS. K.L.H. is supported as a National Science Foundation Graduate Research Fellow. S.L.P. is supported as a Howard Hughes Medical Institute investigator and as a Benjamin H. Lewis chair in neuroscience. This research was supported by the National Institute of Neurological Disorders and Stroke (grant R37NS037116), the Marshall Foundation and the Christopher and Dana Reeve Foundation.
The authors declare no competing financial interests.
Integrated supplementary information
(a) Transverse 50 μm sections of a spinal cord labeled with RabΔG from the gastrocnemius muscle, showing gastrocnemius motorneurons and pre-gastrocnemius neurons from different rostral (top) to caudal (bottom) levels. The lateral propriospinal white matter tracts and the cornu-commisuralis of Marie (CCM) white matter in the dorsal funiculus are indicated. These white matter tracts are known to contain axons of intersegmentally projecting propriospinal interneurons. MSE column neurons can be seen projecting axons into both white matter tracts. (b) Examples of typical cellular morphology of MSE column neurons.
Distribution of spinal neurons pre-synaptic to the wrist extensor, tibialis anterior, hamstrings, and quadriceps motorneurons, each shown at the level of peak motorneurons for that muscle, together with immunolabeling of Tcfap2β (red) and Satb1/2 (white). Images are projected confocal stacks. Scale bar is 250 μm.
(a) Principal component significance plot from principal component analysis on the combined data set of MSE and non-specific ventral location summary metrics (see Methods). The first component (PC1) is the only component identified as significant relative to the significance threshold (dashed red line). (b) PC1 loading pattern represents a reliability signature of low latency to the first motorneuron spike, low standard deviation in the latency to the first motorneuron spike, a high response fraction among a set of ten trials, high similarity in the pattern of motorneuron spikes, and a high fraction of dual root responses with negligible contribution of rostral-caudal location. (c) PC1 association scores (mean and standard error) for MSE evoked L5 (red), MSE evoked L2 (purple), non-specific ventral evoked L5 (orange), and non-specific ventral evoked L2 (blue) responses. A positive association score to PC1 corresponds to greater reliability. (d) Examples of hemisected transverse sections for a transynaptic MSE-focused optical stimulation (left) and a non-specific ventral interneuron stimulation (right). The blue bar represents the dorsal-ventral position of light exposure. MSE and intracord experiments had comparable numbers of ChR2-expressing neurons. Transynaptic (MSE) experiments were only considered if the spinal cords met a minimum criteria for efficiency (at least 15 interneurons in a single peak 50 μm section), and the range was 15-30 interneurons in a peak section in the transynaptic ChR2 experiments. In comparison, intracord injections had an average of 35 ± 10 interneuron per section.
Individual MSE neurons that directly contact two motor pools were identified using RabΔG:GFP that was injected into the medial gastrocnemius and RabΔG:Cherry was injected into the lateral gastrocnemius or quadriceps at P0. Spinal cords were analyzed at P8. The locations (a-c) and high magnification views (d-f) of individual cells that were directly pre-synaptic to the medial gastrocnemius muscle (green) and either the lateral gastrocnemius (red) (a,d) or the quadriceps (red) (b,c,e,f) are shown, as projected confocal images. Double pre-motor MSE are yellow. We found that 13/389 (n=6 cords) pre-medial gastrocnemius neurons were yellow following lateral gastrocnemius or quadriceps injections. We did not observe any yellow cells following injections of the antagonistic pair of medial gastrocnemius and tibialis anterior (0/278), suggesting that yellow double premotor neurons are specifically associated with functionally co-recruited muscles. Scale bars are 250 μm in a-c and 25 μm in d-f.
Projected confocal images of sections from P8 spinal cords with pre-medial gastrocnemius (medial GS-RabΔG, green) and pre-lateral gastrocnemius (lateral GS-RabΔG, cyan) labeling. Excitatory (vGlut2, red) and inhibitory (Gad65 or GlyT2, red) synaptic contacts between medial GS MSE and lateral GS MSE neurons are highlighted with arrowheads. Middle and right panels are single optical sections.
(a-c) In situ hybridization in P10 spinal cords against excitatory vGlut2 (a), inhibitory Gad65 (b), and inhibitory Gad67 (c) in black, with immunolabeling against Tcfap2β (green). (d-e) Immunolabeling in P2 spinal cords against Satb1/2 (red), and inhibitory Pax2 (green) or excitatory Tlx3 (green).
(a) Three-dimensional reconstruction of proprioceptive (PV-syn-Tomato) colocalized terminals (red) onto a RabΔG labeled pre-gastrocnemius MSE neuron (white). (b) Three-dimensional reconstruction of cortical (Ctx-syn-Tomato) colocalized terminals (red) onto a RabΔG labeled pre-TA MSE neuron (white), using Emx1Cre::synaptophysin-tomato. Neurons in a and b express Satb1/2, not shown. Scale bars are 10 μm.
Supplementary Figure 8 Reliability and specificity of distinct motor outputs evoked by optical stimulation of MSE neurons.
L5 (red) and L2 (purple) ventral root responses following 10 trials of optical stimulation of pre-gastrocnemius MSE at L3/L4 (left panel, corresponding to Fig. 6e) and at L2 (right panel, corresponding to Fig. 6d). Note the consistency of the evoked motor patterns. Sets of ten individual traces are shown in each column.
(a) Schematic of experimental setup. Electrodes were placed in the cornu-commisuralis of Marie in the deep dorsal funiculus in densities of RabΔG-labeled MSE axons. (b) Optical stimulation at the site of the rostral electrode (L2) evoked spikes in the local white matter (latency 11.6 ms) preceding those at the caudal site by 1.3 ms. Optical stimulation at the caudal site (c) reversed this relationship. Electrodes were separated by 1.7 mm. The mean conduction delay in n=4 spinal cords was 0.96 ms/mm.
L2 (left) and L5 (right) lumbar spinal cord segments are depicted with the L5 MSE network (orange). This population receives direct corticospinal (purple) and proprioceptive information (red), and indirect inputs from nociceptive sensory pathways (pink). Outputs include motor pools in multiple segments, such as quadriceps (blue, Q) and gastrocnemius (green, GS).
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Levine, A., Hinckley, C., Hilde, K. et al. Identification of a cellular node for motor control pathways. Nat Neurosci 17, 586–593 (2014). https://doi.org/10.1038/nn.3675
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