In order to achieve Objective 1:
i) We used an automated system for operant conditioning to present stimuli on a computer screen (i.e. colours, shapes and sets with different number of dots) and assessed wild-type fish discriminative abilities. Despite fish showing ability to discriminate based on colour, they failed in the shape and numerical discrimination tasks suggesting difficulty in processing complex visual stimuli in the automated set-up.
ii) As fish were unable to perform the numerical task, a more ecological assay was developed. Several studies have suggested the existence of a common magnitude system for representation of both discrete (i.e. number) and continuous quantities (i.e. area). We took advantage of the natural tendency of zebrafish to pass through the larger of two available holes to overcome an obstacle to investigate quantitative skills in this species. Zebrafish showed high accuracy close to humans’ discriminability threshold when discriminating areas and performance was affected by the numerical ratio, in line with literature on both humans and non-human animals. Moreover, no effect of experience was found.
iii) As it has been hypothesized that brain lateralization can increase cognitive efficiency and impact on behaviour, we compared learning of adult zebrafish with left or right behavioural lateralization (measured as left/right turning preference when facing a predator) in a colour discrimination task using an extensive operant conditioning procedure. Anxiety-like behaviour was also assessed by measuring scototaxis response (i.e. preference to move toward darkness). No correlation among behavioural lateralization, scototaxis, and learning skills was found.
iv) However, as behavioural lateralization does not necessarily reflect differences in brain neural circuitry, we then tested learning and impulsivity in fish with different patterns of brain asymmetries, using the 5-choice serial reaction time task. The fish were trained to detect the presence of a briefly presented light in one of 5 holes at one end of the testing tank. During test phase, a variable time delay was introduced before the stimulus presentation. We used premature responses as measure of impulsivity. Data analyses are in progress. However, preliminary results suggest that different degree of brain asymmetry affected learning, impulsivity and anxiety-like behaviour.
To achieve Objective 2:
i) We developed a spontaneous choice paradigm based on the tendency of social species to shoal with the larger group of conspecifics. We found that the ability of wild-type larvae to choose the larger of two shoals increased with age and that the upper limit of their quantitative skills was 2 vs. 5. However, when overall space occupied by conspecifics was equated, no preference was observed thus suggesting that larvae use a combination of numerical and non-numerical features when discriminating between shoals. Fish with different brain asymmetries were also tested. Data analysis is in progress.
ii) The ontogeny of quantitative skills was also investigated using the paradigm developed for adults, where larvae had to pass through pairs of different sized holes. Again, the capacity to discriminate the larger hole decreased as the ratio between the areas increased. There was no difference in performance as a function of age (7, 14, 21 dpf) and the accuracy of larvae almost overlapped that of adults, suggesting a limited role of maturation and experience on the ability to estimate areas.
iii) We also developed a classical conditioning paradigm in which zebrafish larvae, partially restrained in agarose, learned to associate a light stimulus (conditioned stimulus, CS) with a tactile stimulus (unconditioned stimulus, US). resulting in an enhanced tail movement upon exposure to the light in the test phase (conditioned response, CR). The percentage of larvae that learned the association increased with age (range 3-14 dpf). In collaboration with the University of Southern California, the procedure has been automated and will be used to train larvae to learn numerical discriminations.
iv) We used a novel object recognition task in which larvae were initially presented with two identical bi-dimensional objects (T1) and then were exposed to one of the original object and a novel object (T2). Object recognition was defined as a preference for one object over the other in T2, reflecting recollection of the familiar object in T1. Data analyses to assess differences in discriminative abilities and eye preference as a function of brain asymmetries are in progress.
For what concerns Objective 3:
In collaboration with the University of Southern California we developed a novel imaging protocol for whole brain imaging of larvae while assessing numerosities (i.e. 1 and 3 dots changing in size and position). Preliminary results indicated differential neural activation as a function of numerosity suggesting the possible existence of number neurons as previously described in non-human primates and birds.
