The devil is in the detail: Zebrafish learn to discriminate visual stimuli only if salient.

Due to their unique characteristics, the zebrafish plays a key role in the comprehension of neurobiology of cognition and its pathologies, such as neurodegenerative diseases. More and more molecular tools for this aim are being developed, but our knowledge about the cognitive abilities of zebrafish remains extremely scarce compared to other teleost fish. We aimed to investigate the complex cognitive abilities of zebrafish using a tracking-based automated conditioning chamber that allowed precise experimental control, avoided potential cueing provided by the observer (Clever Hans phenomenon), and was shown to considerably improve learning in other teleosts. A computer presented two visual stimuli in two sectors of the chamber, and zebrafish had to enter the correct sector to obtain a food reward. Zebrafish quickly learned to use the conditioning device and easily performed up to 80 trials per day. In Experiment 1, zebrafish efficiently discriminated between two differently coloured sides, reaching a 75 % accuracy in only 10 training sessions. Surprisingly, zebrafish failed to choose the correct chamber when the stimuli were two shapes, a small circle and a small triangle, even when, in Experiment 2, training on shape discrimination was prolonged for up to 30 sessions. In Experiment 3, we tested the hypothesis that simultaneously learning to use the conditioning chamber and learning discrimination imposes a too-high cognitive load. However, zebrafish that first successfully learned how the conditioning chamber functioned (in the colour discrimination) subsequently failed in the shape discrimination. Conversely, zebrafish that firstly failed the shape discrimination subsequently learned colour discrimination. In Experiment 4, zebrafish showed some evidence of learning when the stimuli were two large shapes, suggesting that zebrafish did not discriminate between the shapes of the previous experiments because they were not salient enough. Altogether, results suggest constraints in the discrimination learning abilities of zebrafish, which should be taken into account when developing cognitive tasks for this species.

A psychophysical approach to measuring the threshold for acoustic stimulation in zebrafish (Danio rerio).

Zebrafish are a popular vertebrate animal model for biomedical research including investigations of the auditory system. Responses to acoustic stimulation have been a challenge to carefully measure in zebrafish. Here, the authors have developed a procedure for measuring hearing sensitivity in adult zebrafish using an appetitive automated Go/No Go task. In this task, a trial is initiated when a fish passes through an observing gate. In a sound trial, the fish is reinforced by an automated food delivery system when it enters the reinforcement compartment. If the fish enters the reinforcement compartment during a no-sound trial, a timeout is implemented. Zebrafish successfully learned this task in a median of about ten days of daily training. Zebrafish were most sensitive at a frequency of 800 Hz, which corresponds well with sensitivity reported from physiological methods. As far as the authors know, the present study is the first to provide hearing thresholds for zebrafish using a conventional combination of operant conditioning and psychophysical procedures. This could open the door to other kinds of tests using acoustic stimuli as are commonly conducted in many other laboratory animals.

Differential reinforcement of an approach response in zebrafish (Danio rerio).

Five zebrafish were trained to approach a target using a fully automated training procedure. During a training session, if the distance between the fish and the target was closer than an arbitrarily set distance, the approach response was reinforced by food. The fish continued to respond under this reinforcement contingency and the distance criterion could be shortened up to eighty times within a 1h session. The initial distance limit was then shortened for the next test training session. Once the initial distance criterion was reduced to a final minimum distance, the distance criterion was fixed at this value for the next nine successive sessions. In a second experiment using different fish, we manipulated approach distances in three conditions. The first condition was identical to the changing criterion training as in Experiment 1. In the second condition, only response distances under a distance criterion were reinforced. And in the last condition, only response distances over the distance criterion were reinforced. Results show that zebrafish can control the distance between themselves and a target. In other words, zebrafish are sensitive to the spatial consequences of their behavior. The present results show that a differential reinforcement paradigm can be successfully applied to zebrafish which therefore enhances their value as a vertebrate model for studies of complex behavior including visuomotor learning.

An automated device for appetitive conditioning in zebrafish (Danio rerio).

An automated device and a procedure for the operant conditioning individual zebrafish were developed. The key feature of this procedure was the construction of a simple, inexpensive feeder that can deliver extremely small amounts of food, thus preventing rapid satiation. This allows the experimenter to run multiple trails in a single test session and multiple sessions in one day. In addition, small response keys made from acryl rods and fiber sensors were developed that were sufficiently sensitive to detect fish contact. To illustrate the efficiency and utility of the device for traditional learning paradigms, we trained zebrafish in a fixed ratio schedule where subjects were reinforced with food after 10 responses. Zebrafish reliably responded on the response key for sessions that lasted as long 80-reinforcements. They also showed the traditional "break and run" response pattern that has been found in many species. These results show that this system will be valuable for behavioral studies with zebrafish, especially for experiments that need many repeated trials using food reinforcer in a session. The present system can be used for sensory and learning investigations, as well applications in behavioral pharmacology, behavioral genetics, and toxicology where the zebrafish is becoming the vertebrate model of choice.

Localized brain activation related to the strength of auditory learning in a parrot.

Parrots and songbirds learn their vocalizations from a conspecific tutor, much like human infants acquire spoken language. Parrots can learn human words and it has been suggested that they can use them to communicate with humans. The caudomedial pallium in the parrot brain is homologous with that of songbirds, and analogous to the human auditory association cortex, involved in speech processing. Here we investigated neuronal activation, measured as expression of the protein product of the immediate early gene ZENK, in relation to auditory learning in the budgerigar (Melopsittacus undulatus), a parrot. Budgerigar males successfully learned to discriminate two Japanese words spoken by another male conspecific. Re-exposure to the two discriminanda led to increased neuronal activation in the caudomedial pallium, but not in the hippocampus, compared to untrained birds that were exposed to the same words, or were not exposed to words. Neuronal activation in the caudomedial pallium of the experimental birds was correlated significantly and positively with the percentage of correct responses in the discrimination task. These results suggest that in a parrot, the caudomedial pallium is involved in auditory learning. Thus, in parrots, songbirds and humans, analogous brain regions may contain the neural substrate for auditory learning and memory.

Vocal learning in Budgerigars (Melopsittacus undulatus): effects of an acoustic reference on vocal matching.

Budgerigars were trained to produce specific vocalizations (calls) using operant conditioning and food reinforcement. The bird's call was compared to a digital representation of the call stored in a computer to determine a match. Once birds were responding at a high level of precision, we measured the effect of several manipulations upon the accuracy and the intensity of call production. Also, by differentially reinforcing other aspects of vocal behavior, budgerigars were trained to produce a call that matched another bird's contact call and to alter the latency of their vocal response. Both the accuracy of vocal matching and the intensity level of vocal production increased significantly when the bird could hear the template immediately before each trial. Moreover, manipulating the delay between the presentation of an acoustic reference and the onset of vocal production did not significantly affect either vocal intensity or matching accuracy. Interestingly, the vocalizations learned and reinforced in these operant experiments were only occasionally used in more natural communicative situations, such as when birds called back and forth to one another in their home cages.