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Corticospinal populations broadcast complex motor signals to coordinated spinal and striatal circuits

Abstract

Many models of motor control emphasize the role of sensorimotor cortex in movement, principally through the projections that corticospinal neurons (CSNs) make to the spinal cord. Additionally, CSNs possess expansive supraspinal axon collaterals, the functional organization of which is largely unknown. Using anatomical and electrophysiological circuit-mapping techniques in the mouse, we reveal dorsolateral striatum as the preeminent target of CSN collateral innervation. We found that this innervation is biased so that CSNs targeting different striatal pathways show biased targeting of spinal cord circuits. Contrary to more conventional perspectives, CSNs encode not only individual movements, but also information related to the onset and offset of motor sequences. Furthermore, similar activity patterns are broadcast by CSN populations targeting different striatal circuits. Our results reveal a logic of coordinated connectivity between forebrain and spinal circuits, where separate CSN modules broadcast similarly complex information to downstream circuits, suggesting that differences in postsynaptic connectivity dictate motor specificity.

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Fig. 1: Anatomical characterization of corticospinal neurons.
Fig. 2: Identifying brain-wide inputs to CSNsDLS.
Fig. 3: CSNs are likely to be the only neurons that project to both DLS and spinal cord.
Fig. 4: Optogenetics-assisted mapping of corticospinal collateral synapses in the striatum.
Fig. 5: Mapping the distribution of spinal synapses from CSNsDLS.
Fig. 6: Two-photon calcium imaging of corticospinal neurons during a sequential forelimb behavior.
Fig. 7: Rabies-based in vivo identification of corticospinal neurons with striatal synapses.

Data availability

Data that support the findings of this study are available from the corresponding authors upon reasonable request.

Code availability

Custom code used in this study is available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank K. Fidelin and V. Athalye for feedback on this manuscript. We thank H. Rodrigues for designing and constructing behavioral equipment. We thank S. Brenner-Morton for custom antibodies, and S. Fageiry and K. Ritola for custom viral constructs. We thank Zuckerman Institute’s Cellular Imaging platform for instrument use and technical advice. We are grateful for technical assistance from L. Hammond, G. Martins, M. Correia, C. Warriner, A. Miri and K. MacArthur. We thank I. Marcelo for time-warping code. Imaging was performed with support from the Zuckerman Institute’s Cellular Imaging platform. We thank T. Jessell for inspiring this research and for providing critical feedback. R.M.C. was funded by the NIH (5U19NS104649) and the Simons-Emory International Consortium on Motor Control. A.N. was a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation and is currently supported by a NIH Pathway to Independence Award (1K99NS118053-01).

Author information

Authors and Affiliations

Authors

Contributions

A.N. and R.M.C. designed experiments, interpreted data and wrote the manuscript. A.N. performed experiments and analyzed data. B.A. assisted in collecting and analyzing anatomical tracing data.

Corresponding authors

Correspondence to Anders Nelson or Rui M. Costa.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks Bernardo Sabatini and the other, anonymous, reviewer(s) 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

Extended Data Fig. 1 Mapping brainwide inputs to the spinal cord.

(a) Illustration of approach to visualize inputs to cervical spinal cord. (b) 3D reconstruction of inputs to spinal cord. Colors correspond to major brain divisions. (c) The top brain regions that project to spinal cord, determined by the relative fraction of total somata. Notable regions are indicated by colored bars. Insets illustrate exemplar brain regions with substantial labeling. Dashed boxes are colored to correspond to notable brain regions. The inset pie chart shows the major brain structures (‘major divisions’ as classified by the Allen Institute Mouse Common Coordinate Framework; see Methods) projecting to cervical spinal cord. N = 3. (d-f) Micrographs of CSN axons expressing GFP (green) in transverse cross-sections of cervical (D), thoracic (E), and lumbar (F) spinal cord. The insets are high magnification images of GFP+ bulbous varicosities from different laminae of cervical (7Sp/8Sp), thoracic (7Sp/ICl), and lumbar (4Sp) segments. Neuronal processes expressing Cre.RFP are in red. Representative of N = 3. (g) Quantification of cortical inputs to spinal cord (RFP+), divided by cortical region and laminae. The inset photomicrograph illustrates the L5b positioning of CSNs. Note that the Allen Brain Atlas classification did not subdivide L5 into L5a and L5b, and the position of corticospinal somata fell around the boundary between L5 and L6a. N = 3. (h) Experimental strategy, same as Fig. 1a. (i) The cortical regions giving rise to corticospinal somata (GFP+). Dark green bars represent the major regions; light green bars represent subdivisions of cortical regions. N = 3. (j) Major brain regions containing GFP+ neurites. This includes dendritic processes in sensorimotor cortex. Grouping the many brain regions comprising these major structures reveals the intense innervation of several subcortical structures, including brainstem. (k) Photomicrograph of brainstem labeling by CSNs. N = 3. (l) Experimental strategy to label synapses arising from CSNs. (m) Synaptophysin GFP (green) labeling in the brain. N = 3. (n) Synaptophysin GFP (green) and FlpO (red) labeling in motor cortex. N = 3. (o) Synaptophysin GFP (green) labeling in DLS. N = 3. (p) Top brain regions to which CSNs project, excluding sensorimotor cortex and fiber tracts. N = 2. Error bars are SEM.

Extended Data Fig. 2 Mapping the brainwide targets of CSNsDLS.

(a) Experimental strategy to label corticospinal neurons that project to striatum (CSNsDLS). (b) Photomicrographs exemplifying the cortical distribution CSNsDLS. Representative of N = 3. (c) Sagittal Z projection of raw fluorescence aligned to atlas space. Representative of N = 3. (d) 3D reconstruction of CSNsDLS cell bodies. Representative of N = 3. (e) Quantification of cortical regions contributing to the total population of CSNsDLS, compared to experiments from Fig. 1 targeting primarily the motor cortical population of CSNs (M-CSNs). Asterisks indicate statistically significant differences in innervation between M-CSNs and CSNsDLS (*: p < 0.05, **: p < 0.005, 2-way ANOVA with post-hoc t-test). (f) Quantification of brain regions targeted by CSNsDLS, compared to data from Fig. 1. Note that – despite the differences in experimental strategy – DLS is a primary target of CSNsDLS. N = 3. (g) 3D reconstruction of CSNsDLS projections throughout the brain, colored by targeted brain region. (h) Photomicrograph showing CSNsDLS axon labeling in the brainstem. Representative of N = 3. (i) Higher magnification inset from (H). Representative of N = 3. (j) Experimental strategy to drive expression of GFP in corticospinal neurons that form synapses on identified spinal cell types. (k) 3D reconstruction of axons from CSNsChx10, color coded by brain region. (l) Confocal micrograph exemplifying CSNChx10 axon labeling in DLS. (m) Quantification of brain structures that receive substantial input from CSN subtypes. N = 4, Chx10, N = 2, SST, N = 3, GAD2. Error bars are SEM.

Extended Data Fig. 3 Controls for transsynaptic tracing experiments.

(a) Experimental strategy to confirm Cre-dependent expression of AAVs-FLEX encoding rabies glycoprotein and TVA.mCherry in DLS, as well as dependence of EnVA-N2cΔG-tdTomato infection on expression of TVA. The AAVs were injected into wild type mice, followed by injection of rabies. (b-d) Photomicrographs illustrating the absence of any mCherry or tdTomato labeling in the brain. Representative of N = 3. (e) Experimental strategy to confirm Cre-dependent expression of AAVs-FLEX encoding rabies glycoprotein and TVA.mCherry in the spinal cord, as well as dependence of EnVA-N2cΔG-FlpO.mCherry infection on expression of TVA. (f-g) Photomicrographs illustrating the absence of any mCherry or tdTomato labeling in the spinal cord or brain. Representative of N = 3. (h) Experimental strategy to confirm dependency of transsynaptic spread on rabies glycoprotein, in the DLS. AAV-FLEX-TVA.mCherry was injected into DLS of D1Cre mice. AAV-FLEX-N2cG was omitted from the injection. (i-k) Injecting EnVA-N2cΔG-tdTomato led to local tdTomato expression, but no expression in presynaptic inputs to DLS. Representative of N = 1. (l) Experimental strategy to confirm dependency of transsynaptic spread on rabies glycoprotein, in the spinal cord. AAV-FLEX-TVA.mCherry was injected into spinal cord of GAD2-Cre mice. (m-o) Injecting EnVA-N2cΔG-FlpO.mCherry led to local mCherry expression, but no expression in presynaptic inputs to spinal cord. Representative of N = 3.

Extended Data Fig. 4 Synaptic organization of intratelencephalic corticostriatal projections.

(a) Schematic illustrating the experimental strategy. Retrogradely-transported and expressed AAV encoding ChR2.tdTomato was injected into contralateral DLS or M1. D1 and D2 SPNs were targeted for simultaneous recording. (b) Photomicrograph of ChR2.tdTomato (red) and D2-GFP (green) labeling in a brain slice. (c) High magnification image of the boxed region from (B). Note the expansive axonal plexus. (d) DIC image of a D1+ (magenta) and D1- SPNs targeted for simultaneous whole cell recording. The dashed lines indicate the location of recording electrodes. (e) Superimposed current-clamp voltage recordings from an SPN following optogenetic stimulation of IT corticostriatal axons, highlighting the potency of this projection. (f) Grand average response of all D1 (blue) and D2 (orange) SPNs to optogenetic stimulation of IT corticostriatal neurons. (g-h) Pairwise comparison of ChR2-evoked amplitude (G) and charge (H) in D1 versus D2 SPNs. N = 3 animals, n = 7 cells. Paired t-test. (i-j) Trial average of mEPSCs evoked from an example D1 (I) and D2 (J) SPN. Individual trials are in grey. (k) Average mEPSC amplitudes in D1 versus D2 SPNs. N = 2 animals, n = 7 cells. (l) Distribution of all mEPSCs ordered by mEPSC peak current, recorded in D1 (blue) or D2 (orange) SPNs. The inset is an overlay of the average mEPSC from D1 and D2 SPNs. Error bars are SEM. Shaded areas are SEM. The horizontal dashes in the box plots in (G), (H), and (K) represent the median. The dots in (G) and (H) indicate the means. The bottom and top edges of all boxes indicate the 25th and 75th percentiles, respectively, while the whiskers indicate the minima and maxima, excluding outliers.

Extended Data Fig. 5 Distribution of spinal synapses arising from CSNsDLS.

(a) The same confocal micrograph from Fig. 3b. The ventral horn inset is shown in (b), with vGlut1+ varicosities indicated with arrowheads. (c) Confocal micrograph of immunolabeled calbindin-expressing interneurons located below motor pools in cervical spinal cord (putative Renshaw neurons). (d-g) High magnification 63x single optical section images showing two vGlut1+ synapses from a rabies-labeled CSN in close apposition to the proximal dendrite belonging to a calbindin+ interneuron. (h) Quantification of the mean mediolateral and dorsoventral position of CSNsD1 (blue) and CSNsD2 (orange). N = 5 each, unpaired two−sided t-test, p = 1.08×10−293. (i) Raw CSN spinal synapse data from three example mice. The position of each dot corresponds to a vGlut1+ axonal varicosity. (j) Raw data is spatially binned for each mouse. A sliding window is used to group local bins, and the density of labeling within these groups is compared across genotypes of mice.

Extended Data Fig. 6 Analysis of behavior and deconvolution.

(a) Rate of reward across training. The broken X axis indicates where training changed from Phase 1 (one press issues reward) to Phase 2 (four press sequences issue reward). The green vertical line indicates the day at which the maximum rewarded inter-press interval (IPI) for four lever press sequences is limited to 3 seconds. Before this line (days 8–10), reward was issued every four presses, regardless of IPI. The purple vertical line indicates the day at which the maximum IPI for four lever press sequences to lead to reward is limited to 2 seconds. (b) The inter-press interval of lever press sequences across Phase 2 of training. (c) Confocal micrograph of the spinal cord injection site. (d) Signals extracted from CSNs from one mouse for a portion of a session. Raw fluorescence signal in blue. Calcium signal derived using CNMF is in green. Deconvolved event rate is in orange. Units are a.u. (e) A higher magnification view of signals extracted from one neuron. (f) Histogram depicting the number of deconvolved events as a function of their amplitude. (g) Average of raw fluorescence signal triggered by deconvolved events, aligned to event time. The shade of purple corresponds to the size of the associated deconvolved event. (h) Average of CNMF-derived calcium signal triggered by deconvolved events, aligned to event time. The shade of red corresponds to the size of the associated deconvolved event. Shaded area is SEM.

Extended Data Fig. 7 Imaging the activity of CSNs during behavior.

(a) Trial-averaged calcium activity aligned to lever press for neurons from a single mouse. (b) Same data as (A), but for deconvolved events. (c) Z scored deconvolved events of neurons at baseline versus at lever press. (d) Z scored activity of neurons aligned to single lever press events. (e) Histogram of the times of peak activity relative to lever press, for all neurons. (f) Average activity traces for neurons with peak activity that falls within different bins of time relative to lever press. (g-i) Illustration of time warping procedure for four press sequences. Dots indicate lever press times, as well as time anchors used for pre- and post-trial alignment (six time points per trial). (h) Illustration of how time is either dilated or contracted to match a template sequence. (j-k) Z scored calcium activity before (J) and after (K) time warping, to illustrate the utility of time warping for resolving latent features in unaligned activity. Note the emergence in (K) of peaks in activity corresponding to individual lever press events. This is the same dataset as Fig. 6j, but is calcium signal, instead of deconvolved events. Shaded area is SEM.

Extended Data Fig. 8 Classification of CSN activity during lever press sequences.

(a) The same neurons as (Fig. 6l-n), instead displaying the average of events aligned to first, second, third, or forth press in the sequence. (b) The average activity of neurons belonging to each activity profile, aligned to four lever press sequence onset. (c) Histogram of the times of peak activity for CSNs with categorized activity profiles, aligned to lever press sequence onset. Shaded area is SEM.

Extended Data Fig. 9 Analysis of EMG during behavior and CSN activity correlations to EMG.

(a) Example recording of biceps and triceps muscle activity from one mouse. Biceps EMG is aligned to peaks in triceps EMG. (b) Average EMG activity for four forelimb muscles aligned to single lever presses. (c) Average EMG activity for four forelimb muscles aligned to lever press sequences. (d) Correlation of CSN activity to biceps versus triceps EMG during concatenated random segments of behavior and rest (grey) or concatenated lever press sequences (purple), matched in duration. Paired two-sided t-tests, concatenated random segments: p = 6.66×10−7; concatenated lever press sequences: p = 0.722, N = 8, n = 2252. (e) Correlation of trial-averaged CSN activity with biceps or triceps EMG. Neurons with correlations biased to triceps or biceps are colorized in red or green, respectively. (f) Average lever press sequence-related activity of CSNs highly correlated to triceps (red) or biceps (green) EMG. Activity from neurons with similar correlation coefficients is in grey. (G-L) Analysis of body movements during behavior. (g) A video still with highlighted regions of interest used for quantifying body movements. (h-k) Images of variance from the same vantage as (G) revealing different body parts moving during the behavior. (l) Variance from different body regions of interest aligned to the onset of arm movement sequences. Notice the absence of overt body movements before arm movement. Shaded area is SEM.

Extended Data Fig. 10 Method to identify CSNs with identified striatal synapses.

(a) Exemplar photomicrograph of CSNs expressing GCaMP (green), and corticostriatal neurons marked with tdTomato (red). Representative of N = 8. (b) Cartoon depiction of fluorescent expression possibilities, viewed from an X-Z perspective. (c) Cartoon depiction of fluorescent expression possibilities, viewed from an X-Y perspective. (d) Two example possibilities for overlapping green and red fluorescence, one constituting a double-positive (top) and one rejected from being a double-positive (bottom).

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Nelson, A., Abdelmesih, B. & Costa, R.M. Corticospinal populations broadcast complex motor signals to coordinated spinal and striatal circuits. Nat Neurosci 24, 1721–1732 (2021). https://doi.org/10.1038/s41593-021-00939-w

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