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. 2020 Jun 3;9:e56754. doi: 10.7554/eLife.56754

Figure 1. Functional organization and recruitment order among muscles controlling the Drosophila leg.

(A) Muscles of the right prothoracic leg of a female Drosophila (MHC-LexA; 20XLexAop-GFP). (B) Muscles controlling tibia movement in the fly femur. Top and bottom are confocal sections through the femur. Anterior-posterior axis refers to the leg in a standing posture (Soler et al., 2004). (C) Top: K-means clustering of calcium signals (MHC-Gal4;UAS-GCaMP6f) based on correlation of pixel intensities during 180 s of self-generated leg movements in an example fly (unloaded waving, see methods). Bottom: average change in fluorescence for each cluster, in each frame, when the leg is extended (femur-tibia joint >120°, 20772 frames) vs. flexed (<30°, 61,860 frames), n = 5 flies. Flexion activity was consistently higher (p=0.01 for cluster 5, p<10−6 for all other clusters, 2-way ANOVA, Tukey-Kramer correction). (D) Schematic of the experimental setup. The fly is fixed in a holder so that it can pull on a calibrated force probe with the tibia while calcium signals are recorded from muscles in the femur. (E) Calcium activity in tibia muscles while the fly pulls on the force probe (bottom trace). Cluster six was obscured by the probe and not included. The middle row shows the smoothed, rectified derivative of the cluster fluorescence (dFi/dt) for the two brightest clusters (1 and 2), which we refer to as Flexors. Highlighted periods indicate that both Flexors 1 and 2 are active simultaneously (gray, dF/dt > 0.005), or that Flexor 2 alone is active (magenta). (F) 2D histogram of probe position and velocity, for all frames (n = 13,504) for a representative fly. The probe was often stationary (velocity = 0), either because the fly let go of the probe (F = 0), or because the fly pulled the probe as far as it could (F ~ 85 µN), reflected by the hotspots in the 2D histogram. In F–H), the white circles indicate the centroids of the distributions. (G) Top - 2D histogram of probe position vs. velocity when Flexor 2 fluorescence increased, but not Flexor 1 (n = 637 frames, same fly as F). Gray squares indicate hotspots in F), which are excluded here. Color scaled to log(50 frames). Bottom – example of instance in which Flexor 2 alone is active (magenta shading). (H) Top - Same as G), when Flexor 1 AND 2 fluorescence increased simultaneously (n = 1449 frames). Bottom – gray shading indicates instances of activity in both Flexors 1 and 2. (I) Fraction of total frames for each fly in which both Flexor 1 and 2 fluorescence increased (gray), Flexor 2 alone increased (magenta), or Flexor 1 alone increased. Number of frames for each of five flies: 32,916, 13,504, 37,136, 37,136, 24,476. (J) Shift in the centroid of the 2D histogram when Flexor 1 fluorescence is increasing along with Flexor 2 fluorescence (gray), compared to when Flexor 2 fluorescence alone is increasing (p<0.01, Wilcoxon rank sum test). Black line indicates example cell in G) and H).

Figure 1.

Figure 1—figure supplement 1. Wide-field calcium imaging of muscles in the femur.

Figure 1—figure supplement 1.

(A) Schematic of light path for widefield calcium imaging of femur muscles. Infrared illumination is used to track leg movement. GCaMP emission is reflected by a longpass dichroic mirror to a second camera. (B) Tracking the femur and tibia with DeepLabCut. A network was trained to detect six points on the femur and six points on the tibia in the IR illuminated videos of the leg. We calculated the center of the tibia from the six detected points. The elliptical arc of the centroid across frames allowed us to estimate the angle of elevation of the tibia relative to the plane of the metal holder (black background). (C) K-means clustering of GCaMP activity in muscle fibers during unloaded movements of the tibia. Clusters were similar across five flies, including the ventral distal cluster 1, the proximal cluster 2, the more dorsal cluster 3, and a thin dorsal cluster 4. Clusters 5 and 6 were more variable in shape and location. (D) Example epoch from Fly one showing the femur-tibia angle and the fluorescence of each cluster. (E) Averaged fluorescence across all frames in which the tibia was extended (top) vs. flexed (bottom), for Fly 1, normalized to the maximum ΔF/F. Note the lack of signal during extension. (F) K-means clustering with different numbers of clusters for Fly 1, shown in Figure 1. With fewer than five clusters, the proximal cluster tended to be much larger, incorporating much of the region labeled as cluster 3. With more than six clusters, the smallest and least modulated clusters tended to divide, not providing any further information. When k = 6, pixels in the extension region clustered together, but we did not see large increases in fluorescence with extension (Figure 1).
Figure 1—figure supplement 2. Calibration of the force probe dynamic properties.

Figure 1—figure supplement 2.

(A) The force probe tip was positioned over an analytical balance, and the base of the force probe was displaced. The weight (force) at each displacement is shown in blue, the linear fit is shown in red. The slope of the line is the spring constant, k = 0.2234 N/m. (B) To measure the effective mass and drag constant of the probe in saline, we flicked the force probe by displacing the tip with a glass hook until it lost contact and snapped back to rest. In blue is the displacement in each frame for 16 different flicks. In orange is the velocity. We used the ode45 solver in Matlab to fit the mass (m = 0.17 mg) and drag coefficient (c = 0.14E-3 kg/s). (C) Using the dynamical parameters from b, we calculated the portion of the force due to elastic properties of the force probe (blue), drag (red), and inertia (yellow). Here, a single spike evoked optogenetically in a fast motor neuron produced a small movement of the probe (as in Figure 4A, Video 2). In Figure 4D–F, we calculated force by including drag and inertia, but in other figures we report leg displacement and the approximation of force, assuming that drag and inertia are negligible. (D) The probe obscured the distal leg during spontaneous movements, so we limited the ROI for clustering to the proximal leg.
Figure 1—figure supplement 3. Flexor 2 signal is high whenever Flexor 1 is active.

Figure 1—figure supplement 3.

(A) The force probe histogram for frames when only Flexor 1 was active, scaled according to Figure 1H. (B) Flexor 2 ΔF/F for all frames across N = 5 flies in which 1) Flexor 2 is activating (21962 frames); 2) Flexor 2 alone is activating (6686 frames); 3) when Flexor 1 alone is active (1805 frames); 4) random values of cluster 2 ΔF/F (1805 values). The mean Flexor 2 ΔF/F is higher when Flexor 1 alone is active than when Flexor 2 is active (asterisk, p<10–8, 2-way ANOVA, Tukey-Kramer correction for multiple comparisons), indicating that Flexor 2 is also likely contracting even though ΔF/F is not increasing. (C) Example epochs in which the derivative of Flexor 1 fluorescence, but not Flexor 2 (blue shading) is positive. These epochs make clear that these periods arise when Flexor 2 and 1 are active and contracting together (Flexor 2 ΔF/F high as in Figure 1H), but the derivative of Flexor 2 ΔF/F is either negative or simply not large. The first example occurs near the end of a trial, when the LED was turned off (green arrow).
Figure 1—figure supplement 4. GFP control for k-means clustering of calcium activity.

Figure 1—figure supplement 4.

(A) K-means clustering of GFP expression in muscles during spontaneous movements. Clusters were noisy, interleaved and dispersed. In this case, the fly pulled on the probe and the clusters are superimposed over the image of the fly leg. (B) Changes in fluorescence of clusters calculated from videos of GFP expression during spontaneous movements. GFP expression was bright (as shown in Figure 1A), but did not change with movements.