Fig. 3

Pharmacological analysis of acetylcholine’s role in tentacular contractions. A–D Acetylcholine-induced tentacle contractions in 3-month-old polyps pretreated with 0 µM–10 mM mecamylamine. At 0 µM mecamylamine, 10 mM acetylcholine-induced tentacle contractions in 89 ± 2.32% of animals. This response dropped to 36 ± 10.13% of animals at 1 µM mecamylamine, though not significant (p = 0.095). Acetylcholine-induced tentacle contractions occurred in 20% of animals when pretreated with 10 µM–5 mM mecamylamine and dropped to 0 ± 0% in animals treated with 10 mM mecamylamine (p ≤ 0.05). E–H Nicotine-induced tentacle contractions occurred at 500 µM with 13.33 ± 5.09% and increased to 54 ± 7.99% at 2.5 mM, though these were not statistically different than the controls (p > 0.05). At 5 mM and 10 mM, nicotine-induced tentacle contractions occurred in 100 ± 0% and 98 ± 1.36% of animals, respectively, which was significantly different than the 0 µM–2.5 mM nicotine treatments (p ≤ 0.05). Nicotine-induced complete radial contractions were observed in 38 ± 7.43% of animals starting at 2.5 mM nicotine. At 5 mM and 10 mM nicotine, 70 ± 10.0% and 82 ± 4.05% of animals, respectively had complete radial contractions, these were significantly different than the controls (p ≤ 0.05). I–L Mecamylamine at lower concentrations (0 µM–1 mM) was not capable of blocking tentacular contractions. At 5 mM, nicotine-induced tentacle contractions were reduced to 57.22 ± 16.4%, though not significantly (p = 0.433). At 10 mM mecamylamine, nicotine-induced tentacle contractions were significantly reduced to 3.7 ± 6.8% (p ≤ 0.05). Body contractions were not significantly reduced throughout the mecamylamine gradient, though at high levels (1 mM–10 mM mecamylamine), we did see a trend towards a reduction in the number of body contractions. These data were calculated using a one-way ANOVA (D) Acetylcholine-induced tentacle contractions in 3-month-old polyps pretreated with 0 µM–10 mM mecamylamine F_8,22 = 14.03, p < 0.001; (H, black) Tentacle contraction in the response to nicotine at different timepoints F_7,17 = 43.15, p < 0.001. (H, gray) Body contraction in the response to nicotine at different timepoints F_7,17 = 25.87, p < 0.001. (L, black) Tentacle contraction in 3-month-old polyps pretreated with 0 µM–10 mM mecamylamine F_7,16 = 25.39, p < 0.001; (l, gray) Body contraction in 3-month-old polyps pretreated with 0 µM–10 mM mecamylamine F_6,14 = 3.70, p < 0.05. Each experiment was performed N ≥ 3 times with an n ≥ 7/replicate. Points that do not share letters, either uppercase (in H and L) or lowercase (in D, H, L) are statistically different from each other