Fig. 5. Pharmacology of the action of nicotine in embryonic zebrafish. A, Summary graph indicating that nicotine acts in a concentration-dependent manner. In the rostral spinal cord of 66 hpf islet-1 embryos, ventral myotomes were analyzed to assess whether their motoneuron axons expressed GFP (see Materials and Methods). For example, in the case of controls (Con), of 339 ventral myotomes analyzed in 56 embryos, 291 (85 ± 3%) had axons that expressed GFP. For embryos treated with a 5 μm concentration of nicotine (n = 13 embryos), 84.7 ± 8.7% of the ventral myotomes contained axons expressing GFP; for embryos treated with a 15 μm concentration (n = 31 embryos), 38.3 ± 8.1% of the ventral myotomes contained axons expressing GFP; for embryos treated with a 33 μm concentration (n = 56 embryos), 20.8 ± 4.5% of the ventral myotomes contained axons expressing GFP. *p < 0.0001 (significantly different from control);+p < 0.05 (significant differences between embryos treated with 33 and 15 μm).B, Photomicrographs of a rostral region of spinal cord from a 66 hpf control embryo, an embryo exposed to 33 μmnicotine alone, or an embryo exposed to 33 μm nicotine plus 20 μm DHβE. C, Summary graph of antagonist data in which the analysis was performed as inA. For embryos treated with a 33 μmconcentration of nicotine (Nic) (n = 56 embryos), 20.8 ± 4.5% of the ventral myotomes contained axons expressing GFP; for embryos treated with a 100 nmconcentration of MLA plus a 33 μm concentration of nicotine (n = 13 embryos), 22.0 ± 6.6% of the ventral myotomes contained axons expressing GFP; for embryos treated with a 2 μm concentration of MLA plus a 33 μm concentration of nicotine (n = 11 embryos), 90.7 ± 6.4% of the ventral myotomes contained axons expressing GFP); for embryos treated with a 20 μmconcentration of DHβE plus a 33 μm concentration of nicotine (n = 10 embryos), 84.2 ± 7.9% of the ventral myotomes contained axons expressing GFP. Scale bar, 40 μm. *p < 0.0001 (significance differences compared to 33 μM nicotine exposed).