The limited occupancy of the postsynaptic site by individual axons (see, for example, Figures 1A–1D and 1J) further supports this idea because at most neuromuscular junctions at birth, there is

certainly room for many axons to establish synapses. But this estimate assumes that there is no dominant axon at each junction that occupies a large percentage of the territory, and our calculation is also based on the assumption that the number of innervating axons projecting to the muscle remains constant. We therefore needed to obtain a more direct measure of the number axons converging at neuromuscular junctions at birth. We wanted in addition to assay each of these contacts in terms of its size. Thin-section serial scanning

electron microscopy click here of perinatal neuromuscular junctions provided this information (see Experimental Procedures). Seven hundred serial sections (30 nm in thickness) were imaged in the region of the endplate band and three neuromuscular junctions on adjacent muscle fibers were completely reconstructed (Figure 4A, top panel). Because, as already mentioned, single motor unit labeling showed that axons sent only one branch to each junction they innervated (see Figures 1A–1D), it was possible to count the number of different axons converging at the junction by looking at the number of axons entering the junctional site. We counted 7, 8, and 11 axons entering the three adjacent find protocol junctions (Figure 4 and see Figure S1D available online). In each case, all the axons were bundled in a single fascicle and entered the junctional site from the same direction. All (26/26) of the axons entering the junctions were unmyelinated, although a few myelinated motor or sensory axons were visible in the nerve fascicles coursing through the muscle. To quantify how many of the converging axons were actually establishing synaptic contact with the

underlying muscle fiber, we identified all the sites where vesicle-filled profiles of axons were juxtaposed with the muscle fiber membrane with no intervening glial cell or an open gap of greater than 1 μm. In these three reconstructed junctions, 23/26 these (∼88%) of the axons had sites of contact with muscle fiber membrane (Figure 4A, bottom). The individual terminal arbors of each of the 11 axons innervating one of these junctions are shown in Figure 4B. The three axons that did not have contact with muscle fibers (see, for example, axons 10 and 11 in Figure 4B) terminated in vesicle- and mitochondria-filled bulbs emerging from quite thin axonal branches. Each of the axons that did not contact the muscle fiber was in close proximity to sheathing Schwann cells that contained axosomes (Figure S1C; the yellow-tinted Schwann cell is also shown in panels (ii) and (iv) in Figure 4C).