ZEBRAFISH AS AN INNOVATIVE MODEL TO SCREEN THE BEHAVIOURAL EFFECTS OF NOVEL DRUGS

Zebrafish (Danio rerio) is an emerging animal model alternative to rodents for studying human diseases. Its typical shoaling behaviour (tight aggregation of individuals) consisting of forming a tight group in which fish swim together, may represent an excellent model to study social behaviour. Zebrafish appear to be a good model to study learning and memory, too. The neuropeptides oxytocin (OT) and arginine vasopressin (AVP) are two of the most-studied brain signaling molecules encoding information relevant to social behaviour. Isotocin (ISO) and vasotocin (AVT) are the equivalent neurohypophiseal hormones in fish, regulating reproductive and social behaviour. On this basis, we studied the effect of both OT and AVP in comparison with ISO and AVT, on shoaling, fear response to predator and learning and memory. Social behaviour was studied using mutant zebrafish Nacre. Since these peptides are known to affect anxiety in humans and rodents, the same compounds were also tested on fear response to predator, using Astronotus Ocellatus as stimulus fish. OT (2-40 ng/kg), ISO (0.1-10 ng/kg), AVP (0.5-40 ng/kg) and AVT (0.001-20 ng/kg) were given i.m. 10 min before each test. AVT/AVP were more potent to elicit anxiolytic than social effect while ISO and OT were equally potent. To investigate the mechanism of action, different antagonists were given 10 min before each peptide: the OT receptor antagonist Desgly (0.00001-1 ng/kg), the V1a receptor subtype AVP antagonist SR 49059 (0.00001-20 ng/kg) and the V1b receptor subtype antagonist SSR 149415 (0.00001-1 ng/kg). In both tests, treatment with all the peptides increased social preference and decreased fear response in a dose-dependent manner interpolated by symmetrical parabolas. Pre-treatment with SR 49059, SSR 149415 and Desgly dose-dependently blocked the pro-social and anxiolytic effect induced by each peptide. The less selective antagonist appeared to be SSR 149415. All the neuropeptides did not induce any change in swimming activity. Neuronal nicotinic acetylcholine receptors (nAChRs) play a modulatory role in cognition and zebrafish provide a preclinical model to study these cognitive processes. On the other hand, nicotinic receptor has been characterized in this teleost fish. Using a T-maze task, we investigated the effect of cholinergic drugs on spatial memory in zebrafish. Nicotine (0.0002-0.2 mg/kg), given i.p. 20 min before the test, improved the mean running time difference, showing an inverted U dose-response function. Selective and non selective nAChR antagonists, injected i.p. 10 min before nicotine, were used to study the receptor subunits, involved in spatial memory. Nicotine-induced cognitive enhancement was reduced by the selective nAChR subtype antagonists, MLA (0.01 mg/kg) for α7 subunit, MII (0.1 mg/kg) for α6β2 subunit, DhβE (0.01 mg/kg) for the α4β2 subunit, the non selective antagonist mecamylamine (0.1 mg/kg) and the muscarinic antagonist scopolamine (0.025 mg/kg), with DhβE being more active than MLA or MII. No change in swimming activity was observed for all the nicotinic drugs. Another important cognitive process is the selective attention. It can be assessed in rodents with the novel object recognition (NOR) test. In the standard version of this test, the selection of objects to be used is critical. To overcome the limitation of NOR, we created a modified version of NOR, the virtual object recognition test (VORT) in mice where 3D objects were replaced with stationary geometrical 2D shapes and presented on two Ipods 3.5-inch widescreen displays. A comparable discrimination index as NOR was shown in VORT. 2D shapes that could be highly discriminated and some which could not, were identified. Mice were able to distinguish among different movements (horizontal, vertical or oblique). In fact, the shapes previously found not distinguishable when stationary were better discriminated when moving. Secondly, we focused our attention on zebrafish, which have a good capability to learn and a better visual acuity. Based on this abilities, we investigated in VORT if zebrafish, like mice, were able to discriminate different geometrical 2D shapes (circle, square or triangle), when presented on Ipod-screens, placed at the sides of a water tank. To evaluate the possibility that moving 2D shapes increased the attention of zebrafish, specific movements were applied to the same geometrical shapes. We found that zebrafish, like mice, were able to discriminate different geometrical 2D shapes both stationary and with different movements. In particular, the discrimination index of shapes, previously not discriminate, increased when they were moving. Finally, we investigated if memory performance could be improved by treatment with nicotine both in mice (0.1 mg/kg) and in zebrafish (0.02 mg/kg) or worsened by scopolamine (0.25 mg/kg for mice and 0.025 mg/kg for zebrafish) or by mecamylamine (1 mg/kg). Nicotine improved discrimination index for stationary shapes previously not discriminated while anticholinergic drugs impaired episodic memory in both species. Taken together, these findings showed the pro-social and anxiolytic properties of OT/AVP system mediated by different receptors and confirmed the important role of cholinergic system in the processes of acquisition and memory consolidation in zebrafish similar to mammals. Moreover, we showed, for the first time, both mice and zebrafish could discriminate not only geometrical shapes but also different movements in VORT, allowing a direct comparison between animal model and human to study attention. Zebrafish opens a new avenue of research to rapidly screen new compounds for the treatment of abnormal social behaviours (including autism or schizophrenia) and neurodegenerative diseases.