Figure 2.

TGF-β1 mimics the synaptogenesis effect of SC-CM. A, B, Control nerve–muscle cocultures were treated with trophic stimulation alone. No α-BTX staining was observed at the nerve–muscle contacts (arrowheads) in this example. C, D, Nerve–muscle cocultures were treated with TGF-β1 (10 ng/ml) in addition to trophic stimulation. Note that α-BTX staining was present at nerve–muscle contacts (arrowheads). Scale bar in D applies to A–C. E, The effect of TGF-β1 (10 ng/ml) on the formation of AChR clusters was quantified. Whereas AChR clusters were formed only at 20.3 ± 1.9% of nerve–muscle contacts (n = 985), in cocultures with trophic stimulation only (control, open bar), both TGF-β1 (10 ng/ml) and SC-CM significantly increased the percentage of nerve–muscle contacts associated with AChR clusters (TGF-β1, 59.8 ± 3.3%, 1013 contacts; and SC-CM, 62.3 ± 2.8%, 901 contacts). Altogether, TGF-β1, similar to SC-CM, increased the formation of AChR clusters in nerve–muscle cocultures. F, The dose–response curve of TGF-β1 in the formation of AChR clusters at nerve–muscle contacts. The synaptogenic effect of TGF-β1 ranged from 38.8 ± 1.8% (n = 300 contacts) at 5 ng/ml to 61.9 ± 1.3% (n = 213 contacts) at 20 ng/ml, and it reached a plateau at the concentration of 10 ng/ml (63.2 ± 2.6%, n = 501 contacts). G, The effects of TGF-β1 and SC-CM on the size of AChR clusters at nerve–muscle contacts in nerve–muscle cocultures were quantified. The average size of each AChR cluster formed along nerve–muscle contacts with trophic stimulation alone was 50.5 ± 5.5 μm² (n = 40 contacts) (open bar). The addition of TGF-β1 or SC-CM doubled the size of AChR clusters formed per unit length of nerve–muscle contacts (TGF-β1, 95.4 ± 8.6 μm², n = 121 contacts; and SC-CM, 106.1 ± 7.6 μm², n = 83 contacts). Data are mean ± SEM. Two-tailed, unequal variance Student's t test was used to determine statistical difference. Significance was defined as *p < 0.05.