Effect of BoNT/A on neurotransmitter release of substance P (SP). Off-contractions were recorded at the baseline, in the presence of atropine (1 × 10−6 M), and in the presence of both atropine and CP-96345 (1 × 10−3 M). Compared with boiled BoNT/A-treated muscle, off-contractions were significantly reduced in the BoNT/A-treated muscle. In the presence of atropine, off-contractions dramatically decreased; furthermore, in the presence of both atropine and CP-96345, off-contractions nearly totally diminished. No significant differences existed in the effect of various frequencies of EFS (multivariate ANOVA: effect of BoNT/A treatment, P < 0.05; effect of presence of chemicals, P < 0.05; effect of EFS frequency, P = 0.924). Results were expressed as means ± SE, means of 3 experiments (total N = 12).