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A functional map for diverse forelimb actions within brainstem circuitry

Abstract

The brainstem is a key centre in the control of body movements. Although the precise nature of brainstem cell types and circuits that are central to full-body locomotion are becoming known1,2,3,4,5, efforts to understand the neuronal underpinnings of skilled forelimb movements have focused predominantly on supra-brainstem centres and the spinal cord6,7,8,9,10,11,12. Here we define the logic of a functional map for skilled forelimb movements within the lateral rostral medulla (latRM) of the brainstem. Using in vivo electrophysiology in freely moving mice, we reveal a neuronal code with tuning of latRM populations to distinct forelimb actions. These include reaching and food handling, both of which are impaired by perturbation of excitatory latRM neurons. Through the combinatorial use of genetics and viral tracing, we demonstrate that excitatory latRM neurons segregate into distinct populations by axonal target, and act through the differential recruitment of intra-brainstem and spinal circuits. Investigating the behavioural potential of projection-stratified latRM populations, we find that the optogenetic stimulation of these populations can elicit diverse forelimb movements, with each behaviour stably expressed by individual mice. In summary, projection-stratified brainstem populations encode action phases and together serve as putative building blocks for regulating key features of complex forelimb movements, identifying substrates of the brainstem for skilled forelimb behaviours.

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Fig. 1: Brainstem neurons specifically tuned to forelimb behaviours.
Fig. 2: Excitatory latRM neurons are required for reaching and handling.
Fig. 3: Differential tuning of latRM subpopulation to forelimb behaviours.
Fig. 4: Stimulation of latRM populations elicits specific forelimb movements.

Data availability

Primary data used for analysis in this study are available from the corresponding author upon reasonable request.

Code availability

Custom-made scripts used in this Article are available in a GitHub repository at https://github.com/hk-2019-arber/ruder-et-al-2020.

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Acknowledgements

We thank M. Sigrist, M. Mielich, P. Marini, M. Cases Escuté, P. Capelli and K. Fidelin for experimental help; K. Yamauchi for help with computational analysis of behaviour; L. Gelman and J. Eglinger from the FMI imaging facility and N. Ehrenfeuchter from the Biozentrum imaging facility for help and advice with image acquisition and analysis; J. Courtin (FMI) and members of the Moser laboratories (Trondheim) for advice and help with the acquisition and analysis of the single-unit recordings; P. Argast and P. Buchmann from the FMI mechanical workshop for building devices for behavioural experiments; M. Stadler for help with statistical analysis; and P. Caroni for discussions and comments on the manuscript. All authors were supported by funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Descent, grant agreement no. 692617), the Swiss National Science Foundation, the Kanton Basel-Stadt and the Novartis Research Foundation.

Author information

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Authors

Contributions

All authors were involved in the design of experiments. L.R. together with C.P. carried out most experiments, and acquired and analysed data. R.S. was mainly involved in functional and anatomical experiments related to intra-brainstem interactions, fibre photometry experiments and loss-of-function experiments. H.K. was involved in electrophysiology analysis and loss-of-function experiments. S.V.-G. was involved in early experiments related to intra-brainstem interactions and anatomy. S.A. initiated the project, designed experiments, analysed data and wrote the manuscript. All authors discussed the experiments and commented on the manuscript.

Corresponding author

Correspondence to Silvia Arber.

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The authors declare no competing interests.

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Peer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Methodological approaches and firing properties of latRM neurons.

a, Scheme outlining experimental setup and analysis pipeline for single unit recordings of latRM neurons. A total of 194 neurons were recorded in lever task, pellet task and open field assay. b, Representative latRM section from mouse undergoing single-unit recordings, depicting end point of silicon probe trajectory, visualized through electrical lesion (arrow) performed at the end of all recording sessions, counterstained for ChAT to visualize 7N neurons. c, Analysis of average firing rates of behaviourally relevant neurons for pellet (left, n = 84 neurons) and lever (right, n = 81 neurons) tasks, demonstrating that most neurons fire at relatively low rates. d, Analysis of changes in firing rate of task-tuned neurons comparing baseline to behaviour. The large majority of neurons upregulate their firing rate, and only few downregulate it (n = 43 neurons for lever task, n = 49 neurons for pellet task). e, f, Two examples of raw unsorted traces (e), aligned to reaching (left) or handling (right) onset, depicting the spiking pattern of the subsequently sorted unit below with indication of behavioural time windows. Waveforms for these two units are shown for lever and pellet tasks, which were carried out sequentially. g, Recordings from seven example LatRM neurons during lever or pellet task, displaying single trials aligned to behavioural phases (spikes shown as lines) as well as average firing rate (Hz) below single trials (n = 1 neuron each); grey shade, ±s.e.m.

Extended Data Fig. 2 Behavioural tuning properties of latRM neurons.

ad, Analysis of handling-tuned (a) and lever-reach-tuned (b) latRM populations (n = 34 neurons each), depicting response properties of all respective neurons aligned to behavioural onset of handling, pellet reach, lever reach or locomotion swing phase (top: average of all neurons, bottom: raster plot for individual neurons ordered by peak time of pellet reach) (a) or lever reach (b). c, Data depicted in raster plots for the small number of latRM neurons making up a locomotion swing-phased tuned latRM population. Colour scale in d depicts low (0) to high (1) for relative mean-subtracted firing rate and low (0 Hz) to high (100 Hz) for baseline firing rate. e, Correlation analysis of behavioural tuning of all units analysed in lever, pellet and handling task (n = 5 mice; n = 38 neurons for lever-reach-tuned population, n = 30 neurons for pellet-reach-tuned population (Methods)). f, Summary scheme displaying population cotuning for latRM neurons during lever reaching and pellet reaching. By contrast, the handling-tuned latRM population is not engaged in reaching. Analysed neurons are not tuned to locomotion (swing phase). Grey shades, ±s.e.m.; **P <  0.0025; ***P <  0.00025; Wilcoxon non-parametric signed-rank test. Bonferroni correction was applied to account for multiple comparisons. In a, b, **P <  0.01; ***P <  0.001. Spearman’s rank correlation test (e).

Extended Data Fig. 3 Excitatory latRM neurons are required for precise directional reaching.

a, Experimental scheme for injection of AAV-flex-hM4Di to the latRM of vGlut2cre mice and representative picture of targeting specificity for behavioural experiments, counterstained for ChAT. b, Attenuation of excitatory latRM neurons does not lead to defects in open field locomotion (track length, maximal speed and length of locomotor bouts), comparing PBS and CNO trials (n = 7 mice). c, Quantitative analysis of distance to food pellet, variability and distance to mean, separately shown for PBS and CNO trial days (front camera analysis, same mice as in Fig. 2; n = 7 mice). d, Analysis of point of maximal extension for reaching trajectories using a side camera for recordings (dark coloured circles: average position of trials not missing the target; light coloured circles: same measure for missed trials; each on days with PBS or CNO injection, respectively). e, Quantitative analysis of distance to food pellet, variability and distance to mean, separately shown for PBS and CNO trial days (side camera analysis; n = 7 mice). f, Experimental design for two-choice directional reaching task with lateral and medial reaching positions (left), three examples for recorded latRM neurons (right; n = 1 neuron each), each displaying single trials aligned to behavioural phases (green: reach; yellow: grasp; magenta: retract), spikes shown as lines (top), as well as average firing rates for lateral versus medial recorded trials (bottom). g, Quantification of directionality index (sorted from medial to lateral in ascending order, n = 34 neurons) for latRM neurons recorded during the two-choice directional reaching task. Data are mean ± s.e.m. (grey shades); *P <  0.05; **P <  0.01; two-sided paired t-test.

Extended Data Fig. 4 Excitatory latRM neurons are required for pasta handling but not grip strength.

a, Scheme explaining the approach to quantify pasta angle during handling. b, latRM-hM4Di/DTR-vGlut2 mice do not display defects in grip strength (n = 7 mice hM4Di and n = 3 mice DTR; data are mean ± s.e.m.).

Extended Data Fig. 5 Major synaptic targeting regions of excitatory latRM neurons.

a, Analysis of synaptic output derived from excitatory latRM neurons in vGlut2cre mice to the cervical spinal cord, caudal medulla and contralateral latRM. Representative pictures (left; from one of three mice used for quantification in b) and reconstructions (middle) of SynTag puncta and synaptic density (right) plots for these output structures are shown. Scale bar, 250 μm. b, Quantification of synaptic numbers along the rostro-caudal axis of the cervical spinal cord (C1, C5 and C8). The decrease in synapses between rostral and caudal cervical spinal cord segments demonstrates that spinally projecting excitatory latRM neurons terminate more strongly in rostral cervical spinal cord segments compared to caudal counterparts (n = 3 mice, data are mean ± s.e.m.). c, Summary scheme of main synaptic output areas by excitatory latRM neurons.

Extended Data Fig. 6 Anatomical investigation of rostral medulla neurons on the basis of projections.

a, Example pictures of retrogradely targeted excitatory latRM neurons from cervical spinal cord (from n = 3), MdV- (from n = 2), MdD- (from n = 2) or contralateral (from n = 3) latRM-centric injections counterstained with ChAT (red). Arrows point to cluster of neurons within the latRM, dotted vertical line depicts division between medRM and latRM. Numbers in grey shown in bottom right corner depict percentage overlap for co-injection of two retrograde AAVs into the corresponding output structure. Scale bar, 250 μm. b, Cellular overlap in excitatory latRM neurons retrogradely marked from triple injections in the cervical spinal cord, centred in MdV and in contralateral latRM; representative example shown. There is a minor overlap between the three populations, as indicated by the Venn diagrams (n = 3 mice; dots: position of individual neurons; red dots: overlap with other displayed population; contour lines: density for distribution). c, Analysis of fractions of excitatory rostral medulla neurons residing in medRM versus latRM for four analysed populations shown in different colours (colour code as in Fig. 3, n = 3 mice from triple injections in the spinal cord, MdV-centric and contra latRM, n = 3 mice from MdD-centric), as well as overlap between excitatory medRM neurons retrogradely labelled from the cervical spinal cord and MdV-centric injections (red). d, Experiment combining retrograde targeting of latRM neurons with rAAV-Cre from the spinal cord (left; from n = 3 independent replicates) or contralateral latRM (right; from n = 2 independent replicates) with anterograde injections of AAV-flex-Tomato into ipsilateral latRM. Pictures demonstrate sparse projections of spinally projecting latRM neurons to contralateral latRM (left), and sparse projections of contralaterally projecting latRM neurons to the spinal cord (right), visualizing Tomato immunofluorescence. Scale bar, 250 μm. Data are mean ± s.e.m.; *P <  0.05; **P <  0.01; two-sided paired t-test.

Extended Data Fig. 7 Analysis of activity along the dorsoventral axis in latRM.

a, Experimental scheme depicting recording in dorsal versus ventral latRM during pellet task, with the focus on reaching versus food handling as behaviours (magenta shades: dorsal recording sites; cyan shades: ventral recording sites). b, Pellet-reach-tuned (left; n = 36) and handling-tuned (right; n = 52) latRM population ordered by peak time of respective behaviour onset. Dorsoventral recording position (4 depth) are indicated to the right of plot by a colour code. Bottom plots show average responses of all neurons as well as corresponding shuffled data. Colour scale depicts low (0) to high (1) for relative mean-subtracted firing rate and low (0 Hz) to high (100 Hz) for baseline firing rate; grey shades, ±s.e.m.; ***P <  0.001; Wilcoxon non-parametric signed-rank test. Bonferroni correction was applied to account for multiple comparisons.

Extended Data Fig. 8 Monitoring calcium activity from spinally and MdD-projecting latRM neurons.

a, Fibre photometry data analysing the dynamics of calcium activity in excitatory latRM neurons retrogradely targeted from the cervical spinal cord (n = 4 mice) and from MdD-centred injections (n = 4 mice). Traces are aligned relative to handling onset (dotted line). Shades around mean of individual mice are ±s.e.m. b, Average of mean dynamics of calcium activity for neurons shown in a during onset of locomotion trials (running, n = 4 mice MdD-centred projections, n = 3 mice spinal cord projections) or shuffled data (aligned to reaching onset, n = 4 mice MdD centred projections, n = 4 mice spinal cord projections). Shades around mean of individual mice are ±s.e.m.

Extended Data Fig. 9 Optogenetic activation of rostral medulla subpopulations.

a, Reconstruction of fibre placements and local virus expression sites at the rostral medulla level. Each colour corresponds to one mouse included in the analysis shown in Fig. 4 (code corresponds to mouse ID number shown in Fig. 4d). b, Spatiotemporal analysis of optogenetically induced movements using DeepLabCut. Data depict reaching trajectories (top, left) of different stimulation trials (grey lines) in one mouse (average: cyan), and the lateral view of the trajectory endpoints of reaching mice shown in Fig. 4c using a side-camera (top, right) (Methods). Trajectories of different stimulation trials reconstructed for forepaws ipsi- and contralateral to stimulation during optogenetically-induced tapping (bottom left; average: orange; grey shade, ±s.d.) or grooming (bottom right; average: purple) are also shown.

Extended Data Fig. 10 Stimulation of spinally projecting excitatory latRM neurons recruits forelimb muscles in a sequence resembling natural reaching.

a, Scheme depicting implantation of EMGs into forelimb biceps and triceps muscles, and raw signal demonstrating that these muscles are active in alternation during natural locomotion (below), according to their flexor (biceps) and extensor (triceps) function. b, EMG recordings and quantification (latency and relative onset) for biceps and triceps recordings during optogenetically induced reaching by stimulation of spinally projecting excitatory latRM neurons (top; n = 3 mice for biceps and triceps) or natural reaching (bottom; 0 = reaching onset; n = 3 mice for biceps and n = 2 mice for triceps). Grey shades, ±s.e.m.; *P <  0.05; **P <  0.01; two-sided paired t-test.

Supplementary information

Reporting Summary

Video 1

Chemogenetic attenuation of excitatory latRM neurons leads to directional reaching defects. Representative examples of video sequences showing the behavioral effects of mice expressing chemogenetic inhibitors in excitatory latRM neurons upon PBS (control) or CNO (experimental condition) injection.

Video 2

Attenuation of excitatory latRM neurons leads to pasta handling defects. Representative examples of video sequences showing the behavioral effects of mice handling pasta in mice with functional excitatory latRM neurons compared to loss-of-function experiments either by chemogenetic silencing or DTR-mediated neuronal ablation.

Video 3

Optogenetic stimulation of excitatory RM neurons. Representative examples of video sequences showing the behavioral effects of one mouse for each category upon laser application to stationary freely behaving animals to excitatory latRM neurons projecting the rostral cervical spinal cord, MdD, MdV, or contralateral latRM, as well as excitatory medRM neurons projecting to MdV, and excitatory latRM neurons (n=3 example mice shown).

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Ruder, L., Schina, R., Kanodia, H. et al. A functional map for diverse forelimb actions within brainstem circuitry. Nature 590, 445–450 (2021). https://doi.org/10.1038/s41586-020-03080-z

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