A cembranoid from tobacco prevents the expression of nicotine-induced withdrawal behavior in planarian worms

Abstract

Using an adaptation of published behavioral protocols, we determined that acute exposure to the cholinergic compounds nicotine and carbamylcholine decreased planarian motility in a concentration-dependent manner. A tobacco cembranoid (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R-cembranoid), also decreased planarian motility. Experiments in the presence of 1 microM 4R-cembranoid did increase the IC50 for nicotine- but not carbamylcholine-induced decrease in planarian motility. When planarians were exposed for 24 h to either nicotine or carbamylcholine at concentrations near their respective IC50 values and then transferred to plain media, nicotine-exposed, but not carbamylcholine- or cembranoid-exposed worms displayed withdrawal-like distress behaviors. In experiments where planarians were pre-exposed to 100 microM nicotine for 24 h in the presence of 1 microM 4R-cembranoid, the withdrawal-like effects were significantly reduced. These results indicate that the 4R-cembranoid might have valuable applications for tobacco abuse research. This experimental approach using planarians is useful for the initial screening of compounds relevant to drug abuse and dependence.

Fig. 1

Compounds used in this work. 4R cembranoid (1S,2E,4R,6R,7E,11E-cembra-2,7,11-triene-4,6-diol); (−) nicotine (3-(1-methylpyrrolidin-2-yl)pyridine) and carbamylcholine (2-carbamoyloxyethyl-trimethylammonium).

Fig. 2

A. Motility assay experimental setup (square size=1 cm², see text). B. Effect of 4R-cembranoid in planarian motility. The data points were fit to a linear equation to generate the plots. Each line represents the average of experiments done with 4–10 worms. Similar plots were obtained for nicotine and carbamylcholine (data not shown). Error bars represent the standard error of the mean.

Fig. 3

Dose–response curves showing the effect of the experimental compounds on planarian motility, based on linear plots like Fig. 2B. Each data point represents the average of 4–10 worms. The lines and the IC₅₀ values (μM±SEM) were generated by fitting the data to Eq. (1). Error bars represent the standard error of the mean.

Fig. 4

The presence of 1 μM 4R-cembranoid significantly increases the IC₅₀ of nicotine, but not of carbamylcholine to induce motility decrease in planarians. A. Nicotine. B. Carbamylcholine. The lines and the IC₅₀ s (μM±SEM) were generated using Eq. (1). The p-values were obtained through an F-test. Each data point represents the average of 3–10 worms. Error bars represent the standard error of the mean.

Fig. 5

Nicotine (100 μM, closed symbols), but not carbamylcholine (150 μM, open symbols), induce withdrawal-like behaviors in planarians, as indicated (see methods). Each symbol is the average of three experiments. Error bars are the standard error of the mean. p-values (F-test) are indicated in the figure.

Fig. 6

The “Scrunching/Squirming” behavior does not follow a linear pattern (see text). Nicotine (100 μM, closed symbols), carbamylcholine (150 μM, open symbols). The two data sets are significantly different from each other (p<0.05; two-way ANOVA). Each symbol is the average of three experiments. Error bars are the standard error of the mean.

Fig. 7

Nicotine (100 μM, closed symbols), but not nicotine+4R-cembranoid (100 and 1 μM respectively, open symbols), induce withdrawal-like behaviors in planarians, as indicated (see methods). Each symbol is the average of three experiments. Error bars are the standard error of the mean. p-values (F-test) are indicated in the figure.

Fig. 8

Nicotine (100 μM, closed symbols), but not nicotine+4R-cembranoid (100 and 1 μM respectively, open symbols), induce the “Scrunching/Squirming” behavior (see methods). The two data sets are significantly different from each other (p<0.001; two-way ANOVA). Each symbol is the average of three experiments. Error bars are the standard error of the mean.