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 on the ground. (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 channel of electromyographic (EMG) activity in the long head of the right triceps used to isolate several motor units during walking. (E) Three motor units from the long head identified from the above EMG trace (see Methods). Note that units may only be active in a subset of strides. (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. Note that both (G) and (H) are normalized to total stride duration beginning and ending with elbow minimum extension (blue squares) and show mean (± SE).

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. Each dot reflects an individual motor unit and starred points refer to the examples in (A) and (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 duration across all strides. Black dot within each bar shows 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 while the heavy trace shows the mean and standard error 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. Linear (type 2) regression analysis for the lateral head (R2 = 0.74, p<0.0001) and long head (R2 = 0.70, p<0.0001); regression slopes were significantly different between the lateral and long heads (p<0.01). (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). Linear (type 2) regression analysis for the lateral head (R2 = 0.30, p<0.05) and long head (R2 = 0.42, p<0.05); regression slopes were not significantly different between data from the lateral and long heads.

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 are shown in Supplemental Figure 2.

Motor unit recruitment predicts muscle-specific kinematic differences. (A) We defined elbow extension (Δθ) as the total extension of the elbow over one stride cycle (Δθ = θmax – θmin) on strides in which each motor unit did or did not fire at least one spike (purple tick marks below EMG trace). (B) Each point represents the mean elbow extension observed on strides in which a single motor unit from the lateral (green) or long (purple) head of the triceps fires zero spikes (horizontal axis) vs. when the motor unit fires at least one spike (vertical axis). Stride cycles during which lateral head motor units were recruited (fired at least one spike) exhibited significantly larger elbow extensions than those without spiking (p<0.001, Wilcoxon signed-rank tests), and strides during which long head units were recruited showed significantly smaller elbow extensions than those without spiking (p<0.001, Wilcoxon signed-rank test). Note that in this panel, data for each motor unit were combined across all locomotor speeds. Panel (C) shows the same analysis as in (B), except that 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). This analysis yielded the same result as the one shown in panel (B), with the presence of a spike in the lateral head (green) associated with greater elbow extension and the presence of a spike in the long head (purple) associated with lesser elbow extension (p<0.001, Wilcoxon signed-rank tests). Note that most of the muscle-specific differences shown in (C) were also present when each of the four quartiles were examined individually for each muscle (see Supplemental Figure 3 – supplement to figure 6).

supplement to main text Figure 2.

Empirical observations of spike count distributions for all units. Units are arranged sequentially to match the descending order presented in main text Figure 3.

supplement to main text Figure 5:

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.

supplement to main text Figure 6:

Plots show the same analysis as main text figure 6 for each individual speed quartile. 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.