SC segregation is activity independent but requires axonal presence. (A) Block of neurotransmission by BoTX treatment did not alter morphology of SCs after 3–4 d. Axon (green), synaptic gutter (BTX [red]) and SCs (white [left] and individually pseudocolored [right]). (B–E) Mice with delayed axon fragmentation (ΔNLS) do not show SC intermingling for up to 2 d after transection. (B) Quantification of area overlap after axotomy in ΔNLS mice (mean of SCs + SEM; *, P < 0.01 using a t test; ΔNLS cut, n = 36 SC pairs, three triangularis sterni muscles; ΔNLS uncut, n = 20 SC pairs, three triangularis sterni muscles; WT cut, n = 31 SC pairs, seven triangularis sterni muscles). (C) Single-cell labeling of an NMJ 2 d after axotomy shows segregated SCs and a preserved axon (D) on top of the synaptic gutter (BTX). Despite preserved axon continuity, local axon atrophy was observed (orange arrowheads in D). (E) Time-lapse microscopy over a period of >2 h demonstrates the lack of terminal SC dynamism at axotomized ΔNLS NMJs (shown in the area boxed in C). The timers shown represent hours/minutes. Bars, 5 µm.