Drug classifications of the plant alkaloids. The drug classifications of nicotine, cotinine, anatabine, and nAChR reference compounds (AZD1446 and PNU282987) determined by using the SmartCube^® system in mice are presented. Only nicotine induced anxiolytic-like behavioral signature (yellow bar). Neither the free base nor citrate form of anatabine showed any changes in behavior. Thus, only the free base data are shown for anatabine. The doses are indicated on the x-axis as mg/kg. The color code is described in the figure legend on the left. N = 12 mice.

Effects of alkaloids on anxiety-like behavior in zebrafish. Heatmaps of the general activity of zebrafish after (A) vehicle, (B) nicotine (1 mg/L), (C) cotinine (100 mg/L), or (D) anatabine (10 mg/L) treatment are shown. Nicotine increased the time spent at the top and decreased the time spent at the bottom for the three highest doses, 1, 3, and 10 mg/L (E, H). Cotinine decreased the time spent at the bottom only at 100 mg/L and did not affect the time spent at the top (F, I). Anatabine increased the time spent at the top and decreased the time spent at the bottom at only the highest dose tested (10 mg/L; (G, J). Each individual circle represent one zebrafish. Solid circles = males; open circles = females; n = 12–36; *p < 0.05 and ***p < 0.001. Data are presented as mean ± S.D.

Effects of alkaloids on general movement in zebrafish. Total distance traveled over 5 min test period for (A) nicotine, (B) cotinine, and (C) anatabine and freezing time for (D) nicotine, (E) cotinine, and (F) anatabine are presented. A slight reduction in the movement was detected for fish exposed to nicotine at 1 and 10 mg/L, cotinine at 100 mg/L, and anatabine at 0.3 and 10 mg/L. Freezing time was only increased by 0.3 mg/L anatabine treatment. Each individual circle represent one zebrafish. Solid circles = males; open circles = females; n = 12–36; *p < 0.05 and ***p < Data are presented as mean ± S.D.

FIGURE 4. Effects of nAChR ligands on dopamine release in vitro. Dopamine (DA) release from crude striatal synaptosome preparations were measured after (A) nicotine, (B) cotinine, (C) anatabine, (D) AZD1446 (E) PNU282987, and (F) acetylcholine treatment. All tested compounds elicited robust DA release except for cotinine and PNU282987 at the concentrations tested. Data are presented as mean ± S.D.

FIGURE 5. Effects of nAChR ligands on in vitro norepinephrine release. Norepinephrine (NE) release from crude hippocampal synaptosome preparations were measured after (A) nicotine (B) cotinine, (C) anatabine, (D) AZD1446, (E) PNU282987, and (F) acetylcholine treatment. Only nicotine and acetylcholine seem to elicit clear NE release at the concentrations tested. Data are presented as mean ± S.D.

FIGURE 6. Concentration response curves of the alkaloids for various nAChRs. Functional activity of nicotine, cotinine, and anatabine were tested against (A) α3β4, (B) α4β2, (C) α6/3β2β3, and (D) α7 in vitro. Mean EC₅₀ values (in µM) are indicated in Table 1. Nicotine = black lines with solid circles; Cotinine = grey lines with solid squares; anatabine = grey lines with crosses. Data are presented as mean ± S.D.

Off-target effect assessment. (A) Nicotine, (B) cotinine, and (C) anatabine were tested in 175 in vitro binding or functional assays as summarized in Supplementary Material S2. Each dot represents the outcome of one assay. The grey shaded area covers any assay that showed an effect smaller than 50%. Red dots represent assays that showed greater than 50% change by the respective compounds compared to the vehicle control. N = three repeats.