Fig. 4

Stability of the NMJ postsynaptic domain after nerve injury. a LAL muscles were dissected from control mice and 7, 45, and 90 days after facial nerve resection and processed for immunofluorescence staining to reveal presynaptic motor axons and terminals (magenta) along with BTX to stain postsynaptic densities (cyan). Bar = 40 μm. b Control and nerve injured animals were subcutaneously injected in vivo in the head/neck region with Alexa-488 BTX (cyan, BTX-1). After 7 days, LAL muscles were dissected and incubated with Alexa-555 BTX (magenta, BTX-2). c Following the “two-color BTX assay” NMJs were categorized as “stable” if BTX-1 and BTX-2 labels were similarly intense or “unstable” if BTX-2 intensity was comparatively higher. d LAL muscles from control mice and 5, 7, 8, 9, 10, and 11 days after nerve crush injury were stained for presynaptic motor axons and terminals (magenta) and with BTX to stain postsynaptic densities (cyan). Bar = 20 μm. e, f The NMJ postsynaptic domains from LAL muscles of control adult mice and 10, 14, 21, 37, 60, and 90 days after nerve crush injury were subjected to the “two color BTX assay” and NMJs at different times after nerve crush were categorized as “stable” or “unstable.” The results are represented as the mean ± SEM (N: 3–4 mice; 2 female plus 1–2 male mice). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, Mann–Whitney test. Asterisks show significant differences between stable and unstable structures, as compared to their respective controls