Mature segregated terminal SC territories develop from initial glial intermingling. (A and B) Sequential photobleaching of terminal SCs in P11 young (A) and adult (B) nerve–muscle explants from SC-GFP mice. Sequential bleaching steps are depicted in the middle two panels; subtracted and pseudocolored merges of confocal images are shown in the rightmost panels. (C) Terminal SC area overlap as a percentage of total synaptic (BTX positive) area (young: 11.0 ± 1.5%, n = 30 SC pairs, 10 triangularis sterni muscles; adult: 1.7 ± 0.4%, n = 26 SC pairs, 9 triangularis sterni muscles; *, P < 0.01 using a t test; data are represented as the mean of SC pairs + SEM). (D and E) Terminal SC segregation at young versus mature ages. (D) Three examples are shown: an extensively intermingled young SC pair (8.5% minimum), an intermediate example (36.5%), and a mature highly segregated terminal SC pair (53.7%; maximum segregation index measured). Color-coded dots indicate centroids of individual SCs, and lines indicate the length of the NMJ at the axis through the centroids. (E) Quantification of age-dependent segregation (calculated as the distance of the centroids over the length of the NMJ): young (P7–11; 30.4 ± 1.6%, n = 24 SC pairs, six triangularis sterni muscles) versus adult (44.6 ± 1.3%, n = 23 SC pairs, three triangularis sterni muscles; mean of SC pairs + SEM; *, P < 0.01 using a t test). Bars, 5 µm.