High-resolution muscle recording during mouse locomotion.

(A) (Left) Anatomical landmarks (shoulder, elbow, wrist, paw) and kinematic features (elbow angle θ, paw position x) tracked via high-resolution video during treadmill walking (Darmohray et al., 2019; Mathis et al., 2018). (Right) Position of anatomical landmarks during two stride cycles with limb position captured every 15 ms. (B) Position of the right forepaw (x) relative to body center. Thick lines represent the stance phase when the paw is in contact with the floor of the treadmill. (C) Interior elbow angle (θ) during locomotion. Troughs of this measure, denoting minimum extension (blue squares), were used to define the spike window for each stride. (D) Representative single channel of electromyographic (EMG) activity recorded from the long head of the right triceps. (E) Three motor units recorded from the long head identified from the above EMG trace (see Methods). Note that the bottom-most unit is active in only a subset of stride cycles. (F) Relationship between stride duration and walking speed for all strides in an example mouse. Each dot represents a stride, with shading indicating the speed quartile within which the stride falls (see Methods). (G,H) Right forepaw position x (G) and elbow angle (H) within the walking speed quartiles. Both (G) and (H) are normalized to total stride duration beginning and ending with elbow minimum extension (blue squares) and show mean (± SE).

Motor units identified per muscle in each experimental mouse.

Each thread consisted of 8 electrode contacts used to record bipolar EMG, and numbers in parentheses indicate the number of motor units isolated using the spike sorting algorithm described in Methods. Data from biceps muscle implantation in two mice were not spike sorted and are not included in this report.

Motor unit spike count distributions.

(A) Example motor unit from the long head of the triceps muscle fired zero spikes on 52% of strides, but on the other 48% of strides fired 1-14 spikes. (B) Example motor unit from the triceps lateral head fired zero spikes in 17% of strides but 1-19 spikes on the other 83% of strides. (C) Percentage of strides with at least one spike (probability of recruitment) for all recorded motor units in the long (purple) and lateral (green) heads of the triceps. Symbols denote different animals and each point reflects an individual motor unit. Asterisks highlight the units shown in (A,B).

Motor unit firing patterns within and across muscles.

(A) Example stride with three units from the long head. (B) Mean phase (± SE) of motor unit burst activity within each stride duration across all strides. Black dots on each bar show the mean phase of the unit’s peak firing rate. Starred points refer to the examples in A. (C) Left: Mean time (± SE) between the first spike of a unit’s spike train and the right forepaw footstrike. Positive values denote the spike happening after the footstrike. Right: Mean time (± SE) between the last spike of a unit and the liftoff. Light traces denote values for individual motor units, the heavy trace shows the mean (± SE) across all units within a muscle. (D) Mean peak firing rate (± SE) of each unit. Note that these measurements only include strides in which the given unit was recruited.

Motor unit spike patterns evolve differently in the long and lateral heads.

(A) Relationships between active duration and peak firing rate across motor pools. Type 2 regression slopes were significantly different between the lateral and long heads (p<0.05, permutation test). (B) Motor unit inter-spike intervals (ISIs) across the first three spikes in motor unit bursts. Each data point shows the mean of the first ISI and the ratio between the first and second ISIs for a single unit. Note that by definition only strides with at least three spikes could be used for the analysis shown in panel (B). Type 2 regression slopes were not significantly different between data from the lateral and long heads (p>0.05, permutation test). Data shown here are grouped across all mice; Figure 4-figure supplement 1 shows how these data are distributed across animals.

Motor units alter firing rate and recruitment across walking speeds.

(A) Light traces show median of recruitment probability for individual long head motor units while the heavy trace shows mean (± SE) across all long head motor units. (B) Recruitment probability for lateral head motor units, same plotting conventions as in (A). (C) Difference in recruitment probabilities between slowest and fastest speed quartiles for all motor units. (D) Light traces show median of peak firing rate for individual long head motor units while the heavy trace shows mean (± SE) across all long head motor units. (E) Peak firing rates for lateral head motor units, same plotting conventions as in (D). (F) Difference in peak firing rates between slowest and fastest speed quartiles for all motor units. Across all motor units, both recruitment probabilities (C) and firing rates (F) were significantly higher at the fastest quartile than at the slowest quartile (p<0.01, Wilcoxon signed-rank tests). Speed-dependent changes in recruitment vs. speed-dependent changes in firing rate for individual motor units and experimental animals are shown in Figure –figure supplement 1 and Figure –figure supplement 2, respectively.

Motor unit recruitment correlates with muscle-specific kinematic differences.

(A) We calculated the ranges of elbow angle (Δθ) and elbow velocity (Δω) observed on strides in which each motor unit did or did not fire at least one spike (purple tick marks below EMG trace). (B,D) Each point represents the mean Δθ (B) or Δω (D) observed on strides in which a single motor unit fires zero spikes (horizontal axis) vs. when the motor unit fires at least one spike (vertical axis). Note that in these panels, data for each motor unit were combined across all locomotor speeds. (C,E) Same analyses as before, except each motor unit contributes up to four data points, one for each of the four locomotor speed quartiles in which sufficient data were available (at least 30 strides existed in both the spiking and non-spiking conditions within a given quartile). Legends in each panel show statistical significance for a difference in kinematics tested on the motor units within each muscle (Wilcoxon signed-rank tests). Note that most of the muscle-specific differences shown in (C,E) were also present when each of the four quartiles (Figure 6-figure supplement 1) or experimental animals (Figure 6-figure supplement 2) were examined individually.

Motor unit recruitment probability is greater when other motor units within the muscle are recruited.

Each point reflects a motor unit’s empirically measured recruitment probability, p(MUA), across all strides compared to strides when a simultaneously-recorded motor unit from the same muscle was recruited, p(MUA|MUB). Motor units in each muscle had significantly higher recruitment when another unit was recruited in the same stride (p<0.001, Wilcoxon signed-rank tests).

Isolated motor units had consistent waveforms.

(A,B) Example motor unit waveforms. (Left) Median waveform calculated from a random subset of strides within each trial. (Right) Median waveform calculated from spikes binned in 50ms increments of the stride. (C) (Left) Auto-correlation of each unit’s median waveform between the first trial and subsequent trials. (Right) Auto-correlation of each unit’s median waveform between the first 50ms of its activity and each subsequent 50ms within the stride.

Empirical observations of spike count distributions for all units.

Units are arranged sequentially to match the descending order presented in main text Figure 3. Units 1-17 are in the long head, while units 18-33 are in the lateral head.

Symbols denote different animals.

All other plotting conventions are the same as in Figure 4 in the main text.

Altered firing rate and recruitment across walking speed quartiles for all motor units in the long head (A) and lateral head (B).

Each point reflects the median for the model estimate of each unit across the speeds.

Symbols denote different animals.

All other plotting conventions are the same as in Figure 5 in the main text.

Plots show the same analysis as main text Figure 6 for each individual speed quartile of (A) elbow angle (Δθ) and (B) elbow velocity (Δω).

Speed quartiles 1 and 4 are the slowest and fastest quartiles, respectively, and p-values refer to the results of Wilcoxon signed-rank tests performed separately on data from motor units of the lateral (green) and long (purple) heads of the triceps muscle. Note that most of the muscle-specific differences shown in Figure 6 (C, E) were also present when each of the four quartiles were examined individually for each muscle.

Symbols denote different animals.

All other plotting conventions are the same as in Figure 6 in the main text.