(A) Scheme demonstrating time of tamoxifen treatment and harvest of tissue for P7mTmG mice. (B–C) Representative images of single isolated P7mTmG EDL myofibers from (B) 12-month-old mice, where mGFP is in middle portions where the NMJ is located or (C) 18-month-old mice, where mGFP is located at the end of the fiber. Magnified inset images show NMJs. Scale bar for myofibers = 200 μm, for inset = 25 μm. (D) Quantification of mGFP+ fiber percentage and distribution based on mGFP location (at the end of the fiber or in the vicinity of the NMJ). *p<0.05 for NMJ-associated mGFP compared to 6M/12M groups, #p<0.05 for mGFP at the end compared to NMJ-associated mGFP at that age group, two-way ANOVA/Sidak multiple comparison test. (E) Scheme demonstrating time of tamoxifen treatment and harvest of tissue for Ctrl or P7DTA mice. (F) Confocal IF images and 3-D Amira-based reconstructions (viewed from the pre-synaptic or post-synaptic side) of Ctrl and P7DTA NMJs stained for post-synaptic AChRs (green), nerve terminal markers (red) and nuclei (blue). Post-synaptic myonuclei are indicated with white asterisks, and representative pre-synaptic nuclei indicated with yellow asterisks. Scale bar = 10 μm. (G–H) Quantification of (G) pre-synaptic degenerated, including partially innervated (PI) or completely denervated (Den), or (H) post-synaptic degenerated NMJ percentage of Ctrl and P7DTA TA muscles. *p<0.05, 6M Ctrl/P7DTA, 12M Ctrl vs. 12M P7DTA, 18/24M Ctrl/P7DTA, two-way ANOVA/Sidak multiple comparison test. (I) Percentage distribution of TA NMJs based on number of post-synaptic myonuclei. *p<0.05, 6M Ctrl/P7DTA vs. 12M/18M P7DTA, 24M Ctrl/P7DTA; #p<0.05, 6M Ctrl/P7DTA, 12M Ctrl vs. 12M/18M P7DTA, 24M Ctrl/P7DTA; two-way ANOVA/Sidak multiple comparison test. n = 3 mice per group, ≥50 myofibers examined per mouse (D); n = 3 (18M Ctrl, 24M Ctrl, 24M P7DTA); 4 (6M Ctrl, 6M P7DTA, 18M P7DTA) or 5 (12M Ctrl, 12M P7DTA) mice, >20 NMJs/mouse (G–I).