Behavioral Study of 3- and 5-Halocytisine Derivatives in Zebrafish Using the Novel Tank Diving Test (NTT)

Abstract

Anxiety is a serious mental disorder, and recent statistics have determined that 35.12% of the global population had an anxiety disorder during the COVID-19 pandemic. A mechanism associated with anxiolytic effects is related to nicotinic acetylcholine receptor (nAChR) agonists, principally acting on the α4β2 nAChR subtype. nAChRs are present in different animal models, including murine and teleosteos ones. Zebrafish has become an ideal animal model due to its high human genetic similarities (70%), giving it high versatility in different areas of study, among them in behavioral studies related to anxiety. The novel tank diving test (NTT) is one of the many paradigms used for studies on new drugs related to their anxiolytic effect. In this work, an adult zebrafish was used to determine the behavioral effects of 3- and 5-halocytisine derivatives, using the NTT at different doses. Our results show that substitution at position 3 by chlorine or bromine decreases the time spent by the fish at the bottom compared to the control. However, the 3-chloro derivative at higher doses increases the bottom dwelling time. In contrast, substitution at the 5 position increases bottom dwelling at all concentrations showing no anxiolytic effects in this model. Unexpected results were observed with the 5-chlorocytisine derivative, which at a concentration of 10 mg/L produced a significant decrease in bottom dwelling and showed high times of freezing. In conclusion, the 3-chloro and 3-bromo derivatives show an anxiolytic effect, the 3-chlorocytisine derivative being more potent than the 3-bromo derivative, with the lowest time at the bottom of the tank at 1mg/L. On the other hand, chlorine, and bromine at position 5 produce an opposite effect.

Keywords: anxiety; halogenated cytisine; nicotinic acetylcholine receptors (nAChRs); novel tank diving test (NTT); zebrafish.

Scheme 1

Representative traces of swimming behavior for control (A), 1 mg/L of 3-chlorocytisine (B), and 25 mg/L of 3-chlorocytisine (C). The colors in the traces correspond to their position in the NTT; blue at the top and red at the bottom.

Figure 1

Chemical structure of 3-, and 5-halocytisine derivatives.

Figure 2

Bottom dwelling time (s) upon swimming zebrafish test, using novel tank diving test (NTT). (A) control, 3-chlorocytisine and 5-chlorocytisine (1, 10, 25 and 50 mg/L) groups (n = 10). (B) Control, 3-bromocytisine and 5-bromocytisine (1, 10, 25 and 50 mg/L) groups (n = 10). Ordinary one-way ANOVA with multiple comparisons vs. control. ** p ≤ 0.01 and *** p ≤ 0.001.

Figure 3

Average velocity (cm/s) upon the swimming zebrafish test using the novel tank diving test (NTT). (A) Control, 3-chlorocytisine and 5-chlorocytisine (1, 10, 25 and 50 mg/L) groups (n = 10). (B) Control, 3-bromocytisine and 5-bromocytisine (1, 10, 25 and 50 mg/L) groups (n = 10). Ordinary one-way ANOVA with multiple comparisons vs. control. ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.

Figure 4

Freezing time (s) upon swimming zebrafish test using novel tank diving test (NTT). (A) Control, 3-chlorocytisine and 5-chlorocytisine (1, 10, 25 and 50 mg/L) groups (n = 10). (B) Control, 3-bromocytisine and 5-bromocytisine (1, 10, 25 and 50 mg/L) groups (n = 10). Ordinary one-way ANOVA with multiple comparisons vs. control. * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.

Figure 5

Latency to the top (s) on the swimming zebrafish test using the Novel Tank Diving Test (NTT) (A) control, 3-chlorocytisine and 5-chlorocytisine (1, 10, 25 and 50 mg/L) groups (n = 10). (B) control, 3-bromocytisine and 5-bromocytisine (1, 10, 25 and 50 mg/L) groups (n = 10). Ordinary one-way ANOVA with multiple comparisons vs. control. * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.

Scheme 2

The novel tank diving test (NTT) protocol for testing anxiety behavior. After acclimation in the first holding tanks, the fish was immersed in the drug tank for 3 min and then transferred to the second holding tank before testing in the tank test for 5 min.