Figure 2

Immunohistological analysis of the innervation of the diaphragm and of the morphology of neuromuscular junctions (NMJs). (a) Innervation of the diaphragm by the phrenic nerve, labeled with anti-neurofilament (NF)150-antibody (red), in embryonic wild-type (WT) and knock-out (KO) mice. The phrenic nerve enters the diaphragm (asterisk) and splits up into three primary branches (arrowheads), from which secondary branches (arrow) branch off. Scale bar, 1 mm. (b) Acetylcholine receptors (AChRs), labeled with α-bungarotoxin (α-BTX; green), are clustered on the muscle surface and are innervated by secondary branches of the phrenic nerve in WT and KO diaphragms. Scale bar, 40 μm. (c) Schwann cells, labeled with anti-S100-antibody (red), accompany the phrenic nerve and can be found in close apposition to AChR clusters (green) in WT and KO animals. Scale bar, 40 μm. (d-f) Analysis of the branching pattern of the phrenic nerve in WT and KO diaphragms, which was quantified by determining the number of axon branches that crossed equidistant lines parallel to the main nerve trunk (d). The number of all secondary branches exiting the main nerve trunk on either side (e) and the overall number of bifurcations on either side of the main nerve trunk were also determined (f). Bars represent the mean ± standard error of the mean (SEM) of n = 5 WT or n = 4 KO animals. (g) The number of AChR clusters was determined in diaphragms of WT and KO mice. Bars represent the mean ± SEM of n = 6 WT and 6 KO animals. *P < 0.05.