Translating the behavioural output of the nervous system into movement involves interaction between brain and spinal cord. The brainstem provides an essential bridge between the two structures, but circuit-level organization and function of this intermediary system remain poorly understood. Here we use intersectional virus tracing and genetic strategies in mice to reveal a selective synaptic connectivity matrix between brainstem substructures and functionally distinct spinal motor neurons that regulate limb movement. The brainstem nucleus medullary reticular formation ventral part (MdV) stands out as specifically targeting subpopulations of forelimb-innervating motor neurons. Its glutamatergic premotor neurons receive synaptic input from key upper motor centres and are recruited during motor tasks. Selective neuronal ablation or silencing experiments reveal that MdV is critically important specifically for skilled motor behaviour, including accelerating rotarod and single-food-pellet reaching tasks. Our results indicate that distinct premotor brainstem nuclei access spinal subcircuits to mediate task-specific aspects of motor programs.
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We are grateful to M. Mielich, B. Rapp, D. Salvador and M. Sigrist for expert technical help, L. Gelman, A. Ponti, N. Ehrenfeuchter, M. Kirschmann and R. Thierry for help and advice with image acquisition and analysis, and to P. Caroni for discussions and comments on the manuscript. We thank F. Donato, Y. Zuo, A. Takeoka, P. Tovote and P. Botta for advice on design of behavioral experiments, and S. Sternson for providing PSEM308 and advice for neuronal silencing experiments. M.S.E. was supported by a long-term fellowship of the Human Frontier Science Program, and all authors by a European Research Council Advanced Grant, the Swiss National Science Foundation, the Kanton Basel-Stadt and the Novartis Research Foundation.
The authors declare no competing financial interests.
Extended data figures and tables
Two individual brainstem reconstructions are shown for FL and HL muscle injections. Colour code of brainstem structures and visualization identical to Fig. 1.
Extended Data Figure 2 Differential distribution of forelimb and hindlimb brainstem premotor neurons.
a–f, Direct comparative analysis of distinct FL (purple) and HL (cyan) premotor populations in sagittal ipsilateral (a, c, e) and top-down (b, d, f) view of brainstem reconstructions. Parvicellular reticular nucleus (PCRt) and MdV (a, b), spinal trigeminal nucleus (Sp5) (c, d), and vestibular nucleus (Ve) (e, f) brainstem areas are shown as examples.
a–d, Top-down (a, b) and sagittal contralateral (c, d) views of three-dimensional brainstem reconstructions for FL (a, c) and HL (b, d) premotor neuron analysis. Colour code of displayed neuronal populations is identical to Fig. 1. e, Quantification of neurons contralateral to injected limb (n = 5 mice each). f, g, Pairwise comparison of ipsi- and contralateral premotor brainstem neuron populations by resident nucleus for FL (f) and HL (g) muscle injection.
a–j, Representative reconstructed neuronal morphologies acquired from sagittal sections of gigantocellular reticular nucleus (Gi) (a), magnocellular reticular nucleus (Mc) (b), pontine reticular nucleus (Pn) (c), Raphe (d), parvicellular reticular nucleus (PCRt) (e), spinal trigeminal nucleus (Sp5) (f), spinal vestibular nucleus (SpVe) (g), vestibular nucleus (Ve) (h), and MdV (i; j: from coronal section) are shown in colour according to colour code defined in Fig. 1. Mc cell morphologies showed marked diversity not analysed further in this study. k, Cell soma areas of different premotor populations in the brainstem are displayed (dots represent individual neurons analysed).
a–c, FL premotor neurons in MdV (purple) sparsely overlap with vGATON neurons (a), but colocalize extensively with vGlut2ON populations (b, c) in mice with transgenically marked subpopulations (nlsLacZ, cyan) (n = 4 mice each). d, Quantification of vGATON and vGlut2ON neuron percentages in MdV in relation to NeuN (left) or FL premotor labelling (right) (n = 4 mice each). e, Model illustrating direct connections between vGlutON but not vGATON MdV neurons and FL-innervating motor neurons.
a–f, Top-down (a, b), sagittal ipsilateral (c, d) and contralateral (e, f) views of three-dimensional brainstem reconstructions for biceps (Bic; a, c, e) and triceps (Tri; b, d, f) premotor neuron analysis (n = 5 mice each). Colour code of displayed neuronal populations shown to the right and identical to Fig. 1. g, h, Quantification of neurons ipsi- (g, identical to Fig. 2g for overview purposes) and contralateral (h) to injected limb (n = 5 mice each; ipsi- and contralateral neurons analysed separately). i–n, Direct comparative analysis of distinct Bic (purple) and Tri (cyan) premotor populations in sagittal ipsilateral (i, k, m) and top-down (j, l, n) view of brainstem reconstructions. Sp5 (i, j), Ve (k, l) and Mc (m, n) brainstem areas are shown as examples.
a, Trajectory of descending spinal projections of vGlut2ON MdV neurons marked by coinjection of AAV-flex–Tomato and AAV-flex–Syn–GFP in vGlut2Cre mice. Triple colour immunohistochemistry to Tomato, EGFP and ChAT is shown at C5, C7 and lumbar spinal levels. b, Trans-synaptic rabies spreading from segmentally restricted vGlut2ON or vGATON interneurons at C7-8 levels. Scheme of experimental setup (top left), example pictures depicting MdV neurons connected to vGlut2ON (top middle) and vGATON (top right) spinal interneurons; and visualization of overall distribution (position of triple positive neurons over 3 consecutive sections shown) and example pictures of neurons triple-infected by AAV-TVA/G and EnvA-Rabies viruses in the spinal cord for both experiments (bottom row).
a–c, Experimental setup for analysis of forebrain synaptic input to FL premotor MdV neurons (a) and synaptic input quantification to MdV, Pn and Gi FL premotor neurons (GFPON/vGlut1ON synapse density opposed to premotor neurons; n = 2 mice; b, c). d–g, Example pictures of neurons in paratrochlear nucleus (Pa4) (d), parvicellular reticular nucleus (PCRt) and intermediate reticular nucleus (IRt) (e) connecting to vGlut2ON MdV neurons. Pa4 connecting neurons are not glutamatergic (d). PCRt and IRt neurons are glycinergic or glutamatergic (f, g) as identified by expression of GFP or LacZ in GlyT2GFP or vGlut2Cre transgenic mouse lines.
a, Scheme of experimental setup for c-Fos analysis. b, Quantification of percentage of c-Fos-positive NeuNON (left), vGlut2ON (middle) and vGlut2OFF (right) neurons (dots in graphs represent individual mice). Note that c-Fos is upregulated by motor tasks preferentially in vGlut2ON neurons.
Extended Data Figure 10 Interference with glutamatergic Mc neurons does not perturb single-pellet reaching task performance.
a, Many premotor Mc neurons are glutamatergic (vGlut2ON). b, Specificity of injection site and recombination in Mc of vGlut2Cre mice upon AAV-flex–Tomato injection (py: pyramidal tract). c, Experimental time line for virus injections and task performance of single-pellet reaching task with ablation of glutamatergic Mc neurons. d, Quantitative analysis of different task phases for control (n = 6) and Mc-DTR (n = 6) mice on day 8 of task. Right plot shows FL grip strength analysis.
Visualization of 3D distribution of FL (purple) and HL (cyan) premotor neurons in the brainstem. (MOV 27965 kb)
Visualization of 3D distribution of different subpopulations of FL premotor neurons in the brainstem. Color code for neuronal identity as in Fig. 1. (MOV 23956 kb)
Visualization of 3D distribution of different subpopulations of HL premotor neurons in the brainstem. Color code for neuronal identity as in Fig. 1. (MOV 17756 kb)
Video showing representative examples of different categories for the single pellet-reaching task illustrated in Fig. 5 and 6. Two representative successes, one miss, five no grasp and one drop are shown in sequence. (MP4 13179 kb)
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Esposito, M., Capelli, P. & Arber, S. Brainstem nucleus MdV mediates skilled forelimb motor tasks. Nature 508, 351–356 (2014). https://doi.org/10.1038/nature13023
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