Spatial Distribution of Motor Endplates and its Adaptive Change in Skeletal Muscle

Figure 1. Labeling and clearing of skeletal muscles. (A) Electrophysiological examination by electrical stimulation on the sciatic nerve at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after injecting α-BTX 647 via the tail vein. The amplitude of the CMAP in the gastrocnemius was reduced by over 80% at 60 min after injecting α-BTX 647, and nearly no CMAP was induced in the gastrocnemius at 120 min. (B) Schematic diagrams illustrating MEP labeling by the in vivo injection of α-BTX, and post-cut restaining of the same muscle slices by traditionally incubating in α-BTX 647 solution for 30 min. Notably, α-BTX 647 was a better choice due to the lower background autofluorescence compared to α-BTX 594 (Figure S1). (C) MEPs in the cross-section (100 μm) of the gastrocnemius labeled by an in vivo injection (red) and post-cut restaining using the traditional incubation method (green). Pseudo-colors are used in the images, and both pre- and post-cut labeling were done with the same α-BTX 647. Scale bar: 200 μm. (D) Diagram of sample processing. (E) Transparency of the gastrocnemius before and after 3DISCO clearing. Grid size: 1.45 mm × 1.45 mm. (F) Confocal images of MEPs labeled with α-BTX 647…

Figure 2. Three-dimensional distributions of MEPs in different skeletal muscles. (A-L) Three-dimensional reconstruction of MEPs in the gastrocnemius and quadriceps. A and G show the horizontal view of raw image stacks (white). B and H show the segmentation of muscle surfaces (light gray) and MEPs (red). C and I show the MEPs (red). D, E, F, J, K, and L reveal the cross-sectional view corresponding to A, B, C, G, H, and I. Three-dimensional reconstructions of the tibialis anterior, biceps, and triceps are shown in Figure S5. BS: bone surface; Di: distal end; DS: dorsal surface; Pr: proximal end. Scale bar: 1000 μm. (M-P) The statistical results of different muscles (n = 3 for each muscle): the total number of MEPs (M), volume (N), maximum diameter (O), and number of muscle fibers (P). B: biceps; G: gastrocnemius; Q: quadriceps; T: triceps; TA: tibialis anterior. Data are presented as the mean ± s.d. (Q-T) The correlation analysis between the number of MEPs and each of the following parameters: the number of the head of origins (Q), volume (R), diameter (S), and fiber number (T) of muscles.

Figure 3. Characteristics of MEPs in the denervated gastrocnemius muscles over time. (A) A schematic diagram of the denervated mouse model. (B) Weight of the denervated gastrocnemius relative to healthy muscle. The weight ratio at 2 weeks (W), 1 month (M), 2 M, 3 M, and 6 M after denervation was significantly different from that at 1 W. ***P < 0.001 (one-way ANOVA). Data are presented as the mean ± s.d. (C) Spatial conformation of AChRs stained by α-BTX in MEPs at different time points (0 W, 1 W, 2 W, 1 M, 2 M, 3 M, and 6 M) after denervation. The cross-sectional images in the second row are maximum projections of z stacks (thickness = 250 μm), which are located at the position similar to 0 W, indicated with the yellow dotted box. The enlarged images indicated by white frames are shown in the third row. The scale bars represent 1000 μm for the first row, 500 μm for the second row, and 100 μm for the third row. (D) Band width of the lamella clusters (n = 3, 4, 6, 6, 6 mice for 0 W, 1 W, 2 W, 1 M, 2 M, respectively). n.s.: not significant; **0.001 < P < 0.01; ***P < 0.001 (one-way ANOVA followed by Dunnett's post hoc test) compared with 0 W. Note that there were no obvious MEP lamella…

Figure 4. Distribution of MEPs and the innervation pattern of the gastrocnemius. (A) A schematic diagram of the MIP of the cross-section. (B) MIP of the horizontal-section (thickness = 600 μm). MGL: gastrocnemius lateralis; MGM: gastrocnemius medialis. (C) Resampling cross-sections of the intact gastrocnemius. DS: dorsal surface; TS: tibial surface. (D) Lamella clusters of MEPs in cross-section (thickness = 200 μm) indicated with yellow arrowheads. (E) Gross anatomy of the gastrocnemius and its innervating nerve branches. The gastrocnemius consists of four functional units based on the distribution of MEP lamellas, which are innervated by the medial nerve branch (MNB), the lateral nerve branch 1 (LNB1), the lateral nerve branch 2 (LNB2) and the lateral nerve branch 3 (LNB3), respectively. Grid size: 1.45 mm × 1.45 mm. (F) Segmentation of MEPs (red) and tracing of the nerve branches (green). Scale bar: 500 μm.

Figure 5. Schematic diagram of the functional group based on MEP lamella distribution. Each functional group is formed by one MEP lamella cluster, the corresponding in-muscle nerve branches, and the innervated muscle fibers.