Activity in the motor cortex predicts movements, seconds before they are initiated. This preparatory activity has been observed across cortical layers, including in descending pyramidal tract neurons in layer 5. A key question is how preparatory activity is maintained without causing movement, and is ultimately converted to a motor command to trigger appropriate movements. Here, using single-cell transcriptional profiling and axonal reconstructions, we identify two types of pyramidal tract neuron. Both types project to several targets in the basal ganglia and brainstem. One type projects to thalamic regions that connect back to motor cortex; populations of these neurons produced early preparatory activity that persisted until the movement was initiated. The second type projects to motor centres in the medulla and mainly produced late preparatory activity and motor commands. These results indicate that two types of motor cortex output neurons have specialized roles in motor control.
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We thank A. Lemire and K. Aswath for single-cell sorting and bulk RNA-seq, S. Lindo for stereotaxic surgeries, D. Kao and T. Wang for help with RNA-seq analysis, D. Alcor for imaging assistance, and N. Li and H. Inagaki for help with electrophysiological recordings and for discussion. We thank M. Cembrowski, E. Bloss and F. Henry for discussion. H. Inagaki, M. Sherman, S. Romani, L. Luo, G. Shepherd and T. Wang provided comments on the manuscript. Imaging and reconstructions of neuronal morphology was performed by the Janelia MouseLight project (http://mouselight.janelia.org/). This work was funded by the Howard Hughes Medical Institute, the Allen Institute for Brain Science, the NIH Brain Initiative (U01MH105982 to H.Z.), and the Intramural Research Program of the NIMH (ZIA-MH002497-29 to C.R.G).
Nature thanks P. Carninci, M. Fee, T. Mrsic-Flogel and the anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, b, Heat map of expression (from scRNA-seq) of differentially expressed genes identified from scRNA-seq (a) and bulk RNA-seq (b). Columns represent individual cells, grouped by transcriptomic cluster (indicated below each colour map; Slco2a1, n = 203; Npsr1, n = 43; Hpgd, n = 122) and retrograde labelling site (indicated above). Grey indicates cells isolated from transgenic lines or other targets. Rows represent genes differentially expressed between the Slco2a1 and Hpgd–Npsr1 clusters. Colour bar shows transcript expression in counts per million mapped reads (CPM + 1) on a log-scale. c, d, Heat map of expression (based on bulk RNA-seq) of differentially expressed genes identified from scRNA-seq (c) and bulk RNA-seq (d) datasets. Rows represent genes, coloured by differential bulk RNA-seq expression between thalamus-labelled and medulla-labelled PT neurons. Columns in the heat map represent individual replicates (six each for PTupper and PTlower). Colours show log-transformed transcript intensity (CPM + 1) in z-scored units. Blue shows replicates with low expression (z-score = −2); red shows replicates with high expression (z-score = +2).
a, Axonal lengths of single-neuron reconstructions within targets in thalamus and medulla (thalamus-targeting PT neurons, n = 8, green; medulla-targeting PT neurons, n = 4, magenta). b, Axonal lengths within other selected PT targets. c, Axonal termini in single-neuron reconstructions within thalamic and medullary targets. d, Axonal termini within other selected PT targets. e, Bulk projections of PTupper and PTlower populations. Groups of cortical neurons were labelled from the thalamus (PTupper; mouse #1), the medulla (PTlower; mouse #2), or both targets (mouse #3) using rAAV2-retro expressing spectrally distinct fluorescent proteins. Top, schematics of the labelling procedures. Left, rostro-caudal level (relative to Bregma). Right, annotated coronal sections taken from the Allen Mouse Brain Atlas54 with imaged area indicated. Both cell types extended axon collaterals to motor-related superior colliculus, but to different parts. Axons from PTupper cells were apparent throughout all superior colliculus layers, with a dense projection to the ventrolateral aspect; PTlower neurons were restricted to the ventral superior colliculus and were concentrated more caudally. Both groups project to the pons, particularly the pontine grey, but with terminations in largely non-overlapping zones. PTupper cells projected to the globus pallidus external segment and broadly targeted the dorsal, lateral and ventral striatum. PTlower cells projected sparsely to the lateral striatum. PTlower neurons projected to the central amygdala and parasubthalamic nucleus. PTlower neurons also made up most of the projection to the red nucleus, parabrachial nucleus, substantia nigra pars compacta, motor and sensory trigeminal nuclei in the hindbrain, and through the medullary pyramids. Both cell types extended axon collaterals locally within the same sublamina as their somata, the subthalamic nucleus, zona incerta, and the midbrain reticular nucleus. PTupper cells project more broadly to layer 1 in motor cortex. Mice 1 and 2 were used for electrophysiological recordings. For this reason, projections were labelled with ChR2(H134R)–YFP.
a, The nuclei of PT neurons were retrogradely labelled from the thalamus (green cells) and medulla (magenta cells) using rAAV2-retro. Thalamus-projecting PT neurons are in upper L5b throughout motor cortex, whereas medulla-projecting PT neurons are in deep L5b. Schematics to the left of each image set are annotated coronal sections (Allen Mouse Brain Atlas54). b, rAAV2-retro injection sites in the thalamus (left) and medulla (right). Three biological replicates of this experiment yielded similar results.
Extended Data Fig. 4 Correspondence between scRNA-seq transcriptomic clusters, projection targets, and bulk RNA-seq data.
a, Pairwise correlations (Pearson’s r) in gene expression between all cells in the Slco2a1 cluster and within subsets of Slco2a1 cells identified by projection target. Expression patterns of cells identified from a common target were not more similar than randomly chosen cells. b, As in a for the Hpgd expression cluster. c, Pairwise correlation in gene expression of cells retrogradely labelled from the superior colliculus within the Slco2a1 cluster, within the Hpgd cluster, and between cells from different clusters. Correlations between cells from different clusters were significantly lower than within-cluster correlations (between Slco2a1, superior colliculus and Hpgd, superior colliculus (n = 1,677 pairs) versus within Slco2a1, superior colliculus (n = 903 pairs): P < 1 × 10−10; between Slco2a1, superior colliculus and Hpgd, superior colliculus (n = 1,677 pairs) versus within Hpgd, superior colliculus (n = 741 pairs): P < 1 × 10−10; two-sided Wilcoxon signed rank test). Correlation analysis was not performed for the Npsr1 cluster as the number of cells was relatively small (Fig. 2e). d, Number of differentially expressed genes in bulk RNA-seq data between groups of cells labelled from different projection targets. Notably, only a single gene was identified as differentially expressed between the thalamus/medulla group and the superior colliculus/pons group, indicating that the set of PT neurons projecting to either the thalamus or medulla probably represents a superset of PT neurons. There is unlikely to be a transcriptomically distinct group of PT neurons with projections to the superior colliculus or pons that lacks projections to the medulla and thalamus. Although we cannot rule out a transcriptomically distinct subset that lacks projections to all of the thalamus, medulla, pons and superior colliculus, such neurons were not detected in single-cell reconstructions. e, Total genes detected by bulk RNA-seq and scRNA-seq in PTupper neurons. Both: genes detected in bulk RNA-seq and scRNA-seq; single-cell: additional genes detected only in scRNA-seq; bulk: additional genes detected only in bulk RNA-seq. f, As in e for PTlower neurons. g, Total number of genes detected across all experiments. In e–g, single cell reads were downsampled such that total read depth was the same for scRNA-seq and bulk RNA-seq. h, Number of genes detected in scRNA-seq for each PTupper and PTlower neuron (‘X’, median; PTupper, 9,936 genes; PTlower, 9,865 genes). i, Mean fold change in expression (measured as log2(CPM + 1)) of all genes detected by both methods between neurons in the Npsr1–Hpgd clusters (PTupper) and neurons in the Slco2a1 cluster (PTlower) as determined by scRNA-seq (x axis) and bulk RNA-seq (y axis). Colour represents classification accuracy between the Npsr1–Hpgd and Slco2a1 clusters using a binary (detected/not detected) version of the scRNA-seq data. The 100 most discriminative neurons are coloured for each type. The correlation coefficient (Pearson’s r) in fold change expression was 0.87 for this set of 200 differentially expressed genes.
a, PTupper neurons (n = 61). Left, inter-spike interval histograms; right: trial-averaged activity on lick right (blue) and lick left trials (red). Grey shaded area in inter-spike interval histograms represents the interval of −2.5 ms to 2.5 ms. b, PTlower neurons (n = 69). Boxed region indicates neurons recorded in the right ALM (ipsilateral to injection site in medulla). All other neurons were recorded in the left ALM (contralateral to injection site). c, Bursting cells (cells in which greater than 10% of inter-spike intervals were less than 5 ms) were rare in the PTupper population (3.3%) and more common in the PTlower population (18.8%; P = 0.006, Fisher’s exact test).
a, Green line: variance of trial-averaged activity explained by increasing numbers of principal components across the population of PTupper neurons (n = 61). Dark green line and 95% confidence interval: expected variance explained by the same number of components for size-matched samples (n = 1,000 repetitions) of simultaneously recorded, unidentified neurons. b, Ratio of variance explained between PTupper neurons and bootstrapped distribution of simultaneously recorded unidentified neurons. Error bars represent s.d. of distribution. c, d, As in a and b, but for PTlower neurons (n = 69). Asterisks denote points significantly greater than unity (P < 0.05, bootstrap).
a, Time course of the linear combination of neuronal activity that best differentiates trial types in the 400 ms immediately before the go cue (late coding direction; CDlate) on lick right (blue) and lick left (red) trials for PTupper (top; n = 61) and PTlower (bottom; n = 69) neurons. b, Difference in CDlate projections on lick right and lick left trials (selectivity) in each population. Selectivity along CDlate is present in both populations, and persists after the go cue, but is not strongly modulated during movement initiation. Shaded regions represent the s.d. of the distribution produced by hierarchical bootstrapping (n = 1,000 iterations) in a and 5–95% confidence intervals in b (denoting region significantly greater than zero, P < 0.05 one-sided test, bootstrap). c, A coding direction (CD) was calculated at all individual time points. Heat maps represent the correlation (inner product) of the CD between pairs of time points. In PTupper neurons (top), the coding direction remained similar across the sample and delay epochs. In PTlower neurons (bottom), coding directions in the delay epoch were largely orthogonal to coding directions calculated in the sample epoch. The upcoming movement direction is encoded in a persistent manner in the PTupper population, but not the PTlower population. Right, expanded view of the change in coding direction around the time of the go cue. An abrupt change in the coding direction occurs immediately after the go cue onset in the PTlower population. A change also occurs in the PTupper population, but more slowly (over several hundred milliseconds), largely after initiation of movement. d, Correlation (Pearson’s r) between CDearly vector weights and CDlate vector weights for PTupper neurons (top) and PTlower neurons (bottom). e, Correlation between the CD and CDlate normalized to the mean correlation in the 400 ms preceding the go cue. A rapid change in the CD occurs in the PTlower population following the go cue. Shaded areas represent the s.d. of the bootstrapped distribution (n = 1,000 iterations).
Extended Data Fig. 8 Comparison between identified PT populations and simultaneously recorded untagged neurons.
Time course of selectivity along CDearly (as in Fig. 5b), CDgo (as in Fig. 6b), and CDgo aligned to the last lick in each trial (as in Extended Data Fig. 10b) calculated as follows: (1) from PTupper (n = 61) and PTlower (n = 69) neurons (top row), (2) from simultaneously recorded but unidentified neurons (second row; n = 495 from PTupper cohort; n = 511 simultaneously recorded with PTlower neurons), (3) after removing unidentified neurons from the PTlower experiments recorded from the contralateral hemisphere (third row; n = 276 remaining), which were recorded at a lower average depth in a different recording configuration, and (4) from PTupper and PTlower neurons with firing rates z-scored based on their firing rates in the epoch preceding the stimulus (bottom row). CD projections in the populations of unidentified neurons were similar and nearly indistinguishable after they were approximately depth-matched. Shaded regions represent 5–95% confidence intervals in bootstrapped distribution as in all other figures (n = 1,000 iterations; denotes region significantly greater than zero, P < 0.05 one-sided test, bootstrap). b, Correlation of CD with CDlate (inner product; as in Extended Data Fig. 7e) around the time of the go cue in each PT population (left), in simultaneously recorded unidentified populations (middle) and approximately depth matched populations (right). The change in correlations around the time of the go cue in the unidentified populations were similar and intermediate between that observed in the PTupper population and PTlower populations. c, Early trial type decoding (as in Extended Data Fig. 9) in each PT population (left) and the simultaneously recorded populations of unidentified neurons (right).
a, Accuracy of trial type classification by single neurons in the 400 ms immediately after stimulus onset. 24.6% (15 out of 61) of PTupper neurons predicted trial type with at least 70% accuracy, whereas only 4.4% (3 out of 69) of PTlower neurons did so. Mean accuracy was also significantly higher in PTupper neurons (PTupper: 64.4 ± 1.0%; PTlower: 58.9 ± 0.6%, mean ± s.e.m.; P = 1 × 10−4, two-sided Mann–Whitney test). The ten most discriminative neurons all belonged to the PTupper population. b, Cumulative distribution function of the data in a. c, d, As in a and b but decoding only based on spike rates rectified at baseline. Trial-type selectivity during the sample epoch in PTlower neurons was predominantly characterized by a modest suppression of spiking on one trial type, probably reflecting widespread lateral inhibition. Disregarding spike rate changes below baseline, no PTlower neurons predicted trial type with at least 70% accuracy, whereas the same 24.6% of PTupper neurons continued to do so and accounted for 20 out of 21 of the most predictive neurons (PTupper: 62.7 ± 1.1%; PTlower: 56.7 ± 0.4%, mean ± s.e.m.; P = 9 × 10−7, two-sided Mann–Whitney test). As soon as the trial type is cued by the stimulus, upcoming movement direction is encoded robustly in a subset of PTupper cells and only minimally in PTlower cells.
a, Selectivity along CDlate (as in Extended Data Fig. 7b) for PTupper (green, left; n = 61) and PTlower neurons (magenta, right; n = 69) aligned to the last lick in the response epoch. b, Selectivity along CDgo (same as Fig. 6b) aligned to the last lick for each PT type. Shaded regions in a and b represent 5–95% confidence intervals around the mean using hierarchical bootstrapping (n = 1,000 iterations; denoting region significantly greater than zero, P < 0.05 one-sided test, bootstrap). c, Correlation of coding direction weights at all pairs of time points after the go cue for PTupper neurons (left) and PTlower neurons (right) using last-lick aligned spike rates. An additional transition in the population dynamics accompanies the termination of movement in PTlower neurons, whereas there is no correlate of movement termination in PTupper neurons. The change in dynamics at the offset of movement was less abrupt than at movement onset, probably a result of aligning data to the last lick-port contact, which does not precisely mark the cessation of movement. d, Spike raster plots (top) and trial-averaged activity (bottom) for four example PTlower neurons aligned to the go cue (lick right: blue; lick left: red) and the last lick-port contact (lick right: dark blue; lick left: dark red).