The comparison of the chemical structures of nicotine and nicotinic derivatives: cotinine and 6-hydroxy-L-nicotine (CYP2A6—Cytochrome P450 2A6; NDH—nicotine-dehydrogenase).

The timeline and experimental design of the study (IMP-imipramine; GAL-galantamine; AD-Alzheimer’s disease; SCOP-scopolamine; NIC-nicotine; COT-cotinine; 6HLN-6-hydroxy-L-nicotine).

The effects of nicotine (NIC), 6-hydroxy-L-nicotine (6HLN), and cotinine (COT) (1 and 2 mg/L) administration in scopolamine (SCOP)-treated zebrafish on anxiety-like behavior and locomotor activity evaluated within the novel tank diving test (NTT). (A) The zebrafish locomotion tracking pattern in the NTT according to the experimental groups they belong to. The blue dot represents the beginning of the route while the red dot represents the end of the route. The distance travelled in the top (B) and the time spent in the top/bottom (C) were endpoints for measuring the anxiety, whereas the total distance travelled (D) and the velocity (E) were endpoints for measuring the locomotor activity. The values are expressed as means ± S.E.M. (n = 10). ANOVA analysis identified overall significant differences between the experimental groups for (B) F(8,81) = 21.35, p < 0.0001, (C) F(8,162) = 42.29, p < 0.0001, (D) F(8,81) = 8.555, p < 0.0001 and (E) F(8,81) = 6.172, p < 0.0001. For Tukey posthoc analyses—**** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05.

The effects of nicotine (NIC), 6-hydroxy-L-nicotine (6HLN), and cotinine (COT) (1 and 2 mg/L) administration in scopolamine (SCOP)-treated zebrafish on spatial memory and locomotor activity evaluated within the Y-maze task. (A) The representative locomotion tracking pattern of the zebrafish in the second stage of the Y-maze task according to the experimental groups they belong to. The arms of the maze were denoted with A (start arm), B (other arm), and C (novel arm). The blue dot represents the beginning of the route, while the red dot represents the end of the route. The time in the novel arm (B) represents a parameter for spatial memory, whereas the (C) total distance travelled and the (D) turn angle is measures of locomotor activity. The values are expressed as means ± S.E.M. (n = 10). ANOVA analysis identified overall significant differences between the experimental groups for (B) F(8,81) = 10.54, p < 0.0001, (C) F(8,81) = 5.342, p < 0.0001 and (D) F(8,81) = 8.96, p < 0.0001. For Tukey posthoc analyses—**** p < 0.0001, *** p < 0.001, ** p < 0.01and * p < 0.05.

The effects of nicotine (NIC), 6-hydroxy-L-nicotine (6HLN), and cotinine (COT) (1 and 2 mg/L) administration in scopolamine (SCOP)-treated zebrafish on recognition memory evaluated within object discrimination task. (A) The locomotor tracking pattern of the zebrafish within the testing session of the object discrimination task according to the groups they belong to. The blue dot represents the beginning of the route, while the red dot represents the end of the route. The preference percentage (B) was considered the endpoint for recognition memory. The familiar and the novel objects were noted with F and N, respectively. The black dashed line (chance) indicates a 50% preference. The values are expressed as means ± S.E.M. (n = 10). ANOVA analysis revealed overall significant differences between groups for (B) F(8,81) = 8.078, p < 0.0001. For Tukey posthoc analyses—**** p < 0.0001, and ** p < 0.001.

The effects of nicotine (NIC), 6-hydroxy-L-nicotine (6HLN), and cotinine (COT) (1 and 2 mg/L) administration in scopolamine (SCOP)-treated zebrafish on acetylcholine esterase (AChE)- (A), superoxide dismutase (SOD)- (B), catalase (CAT)- (C) and glutathione peroxidase (GPX)- (D) specific activities and the content of glutathione (GSH) (E), malondialdehyde (MDA) (F) and carbonylated proteins (G). The values are expressed as means ± S.E.M. (n = 3). ANOVA analysis revealed overall significant differences between the experimental groups for (A) F(7,16) = 11.5, p < 0.0001, (B) F(7,16) = 10.15, p < 0.0001, (C) F(7,16) = 11.31, p < 0.0001, (D) F(7,16) = 14.22, p < 0.0001, (E) F(7,16) = 8.541, p < 0.001, (F) F(7,16) = 12.39, p < 0.0001 and (G) F(7,16) = 14.53, p < 0.0001. For Tukey posthoc analyses—**** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05.

Effects of nicotine (NIC), cotinine (COT), and 6-hydroxy-L-nicotine (6HLN) (1 and 2 mg/L) administration on brain-derived neurotrophic factor (bdnf) (A), neuropeptide Y (npy) (B), early growth response protein 1 (egr1) (C), and nuclear factor erythroid 2-related factor 2 (nrf2a) (D) gene expression. The values are expressed as means ± S.E.M. (n = 4). ANOVA analysis identified overall significant differences between groups for (A) F(7,24) = 16.65, p < 0.0001, (B) F(7,24) = 14.79, p < 0.0001, (C) F(7,24) = 19.87, p < 0.0001 and (D) F(7,24) = 32.96, p < 0.0001. For Tukey posthoc analyses—**** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05.

Pearson’s correlation coefficient between behavioral or biochemical parameters and malondialdehyde (MDA) or markers of gene expression (n = 10 animals per group): (A) Time in the novel arm (% of total time) vs. early growth response protein 1 (egr1) (r = 0.648, p < 0.001), (B) Preference % vs. neuropeptide Y (npy) (r = 0.421, p < 0.05), (C) Acetylcholine esterase (AChE) vs. malondialdehyde (MDA) (r = 0.819, p < 0.001), (D) Catalase (CAT) vs. malondialdehyde (MDA) (r = −0.613, p < 0.001), (E) Catalase (CAT) vs nuclear factor erythroid 2-related factor 2 (nrf2a) (r = 0.546, p < 0.001) and (F) The content of glutathione (GSH) vs. nuclear factor erythroid 2-related factor 2 (nrf2a) (r = 0.678, p < 0.001) in control (■), scopolamine (SCOP) (X), nicotine (NIC) 1 mg/L + scopolamine (SCOP) (▼), nicotine (NIC) 2 mg/L + scopolamine (SCOP) (▲), 6-hydroxy-L-nicotine (6HLN) 1 mg/L + scopolamine (SCOP) (●), 6-hydroxy-L-nicotine (6HLN) 2 mg/L + scopolamine (SCOP) (♦), cotinine (COT) 1 mg/L + scopolamine (SCOP) (►) and cotinine (COT) 2 mg/L + scopolamine (SCOP) (◄). Data are expressed as follows: AChE (nmol ATCh/min/mg protein), MDA (µmol/L), CAT (U/mg protein), GSH (µg GSH/µg protein), nrf2a (mRNA copy number, ×10,000) and egr1 (mRNA copy number, × 10,000).