11,2-bis-N-cytisinylethane (CC4), a new nicotinic partial agonist, blocks nicotine- and Δ-9-tetrahydrocannabinol-induced conditioned place preference in zebrafish

Nicotine and cannabis abuse is widespread in Western societies, especially among teenagers and young adults, and a number of findings suggest that cholinergic and endocannabinoid systems interact in modulating reward-related processes [1]. In order to introduce new therapies to reduce the psychotropic effect of nicotine and cannabis, it is of primary importance to discover new pharmacological tools, and the cholinergic system seems to be a possible target. CC4 is a dimer of cytisine that lacks the latter’s agonist properties but potently antagonises or partially and not very effectively agonises various nAChR subtypes. As the in vivo activity of this new compound has not yet been investigated, we analysed its effects in Zebrafish (Danio rerio), an emerging animal model for the study of addictive behavior [4] in which neuronal nicotinic receptors have been identified [5]. The aim of this study was to evaluate the ability of CC4 to interfere with the conditioning place preference (CPP) induced by nicotine and THC. Each fish was placed in a see-through tank divided into two compartments with distinct visual cues. Nicotine (0.0001-0.1 mg/kg) or THC (0.0075-6 mg/kg) was administered intramuscularly immediately before the tests, and CC4 (0.01-10 mg/kg) was given alone or in combination with the maximal reinforcing dose of nicotine or THC. Nicotine and THC induced CPP in a dose-dependent manner, but pretreatment with CC4 fully reversed the maximal reinforcing effects of both drugs. The observed rewarding effects were specific because there was no change in swimming behaviour. In order to characterize the nicotinic receptors in the zebrafish, we performed binding experiments using radio-labeled nicotinic ligands and identified two distinct classes: high-affinity  bungarotoxin receptors and high-affinity epibatidine receptors. We are currently characterizing the two classes pharmacologically and biochemically. Our findings suggest that zebrafish is a useful model for studying drug addiction and identifying new compounds (such as CC4) for the possible treatment of addiction.