Dependence of nicotine adaptation on PKC.A, Response of tpa-1 mutants to levamisole. Levamisole dose–response curves for wild-type (N2) andtpa-1 mutant animals are shown. Animals were assayed for 4 hr under the indicated condition; points and error bars indicate the mean and SEM of at least 12 trials. B, Defect of tpa-1 mutants in nicotine adaptation. Levamisole dose–response curves (assayed at 4 hr) for wild-type andtpa-1 mutant strains after overnight treatment with 30 mm nicotine are shown. Points and error bars indicate the mean and SEM of at least 12 trials. The responses of bothtpa-1 mutants were significantly greater than that of the wild-type under this condition according to the Mann–Whitney rank-sum test (*p < 0.001). By the same test, thetpa-1 mutants showed no significant reduction in response to levamisole after overnight nicotine treatment (p > 0.5). C, Effect of nicotine on vulval muscle UNC-29 protein levels in tpa-1mutants. Shown here are digital images of naive (left) and nicotine-treated (right) tpa-1 mutant hermaphrodites expressing UNC-29:: GFP in the vulval muscles under the control of the myo-3 promoter (see Fig. 4).Long arrows indicate vulval muscles, and ashortarrow indicates the vulva. Animals of the strain AQ521 (genotype, unc-29(x29); tpa-1(k501); Ex [pmyo-3:: unc-29:: GFP; rol-6d]) were cultured on NGM containing 30 mm nicotine overnight; UNC-29:: GFP fluorescence was measured as described previously. D, Quantification of UNC-29:: GFP and SNB-1:: GFP fluorescence in nicotine-treated and untreated animals. The average intensity of fluorescence for nicotine-treated and naive animals expressing UNC-29:: GFP protein under the control of the unc-29 promoter (unc-29:: GFP) or themyo-3 promoter (myo-3:: unc-29:: GFP) or the synaptic marker VAMP:: GFP under the control of the VC-specific unc-4 promoter (punc-4:: snb-1:: GFP) are shown. Asterisks indicate significantly less fluorescence after nicotine treatment according to the Mann–Whitney rank-sum test (*p < 0.001). The histogram and error bars represent the mean and SEM of five trials.