# How to Make Your Fish Work for You: Tips from Ethology and Ecology for Finding Appropriate Unconditioned Stimuli for Learning Studies with Zebrafish

## TL;DR

This paper discusses how to better motivate zebrafish for learning studies by considering their natural behaviors and ecology.

## Contribution

The paper introduces the idea that understanding zebrafish ethology and ecology can improve the design of learning and memory experiments.

## Key findings

- Making zebrafish hungry can paradoxically reduce their motivation to learn about food.
- Food may be an ineffective unconditioned stimulus due to zebrafish foraging behavior.
- Alternative stimuli like conspecifics or ecologically relevant cues may be more effective.

## Abstract

The zebrafish is gaining popularity in a variety of subfields of biology. One of these rapidly evolving fields is behavioural neuroscience. In particular, for the analysis of learning and memory, the zebrafish has been proposed as an alternative to traditional rodent models. Although zebrafish have been demonstrated to be able to learn and remember, perhaps due to the publication bias against non-significant results, failed learning and memory studies, including our own, remained unreported. In this perspective article, I argue that an important, and unresolved, problem may have contributed to these failures: our lack of understanding of how to properly motivate zebrafish. I illustrate the issues using studies conducted in my own laboratory. I discuss paradoxical findings, e.g., why making zebrafish hungry reduces their motivation to learn to find food. I also elaborate on how borrowing from nature could help us find better ways to motivate zebrafish and thus design more appropriate learning and memory tasks. I also discuss the deleterious effects of confounding factors, including those of human handling, and briefly mention potential ways to minimize such effects. Despite the above complexities, I present an optimistic view for the future of zebrafish in both basic and translational research.

A key requirement of associative learning studies is the ability to motivate the subject to acquire memory of the conditioned stimulus–unconditioned stimulus (CS–US) association. Although zebrafish have been found capable of acquiring CS–US associative memory, in many studies, the fish failed to learn. One reason for the failure, I argue in this perspective article, is that we do not yet know how to motivate zebrafish. I illustrate this problem using examples, and offer some solutions, based upon results obtained in my own laboratory for appetitive associative learning tasks for zebrafish. I highlight the value of considering the ethology and ecology of the zebrafish. I discuss why food may have been an ineffective US for zebrafish. I provide examples for how to improve the rewarding properties of food based upon the foraging behaviour of zebrafish in nature. I discuss the efforts to identify alternative USs, including the sight of conspecifics or the presence of other ecologically relevant stimuli. I theorize about conflicting motivators in zebrafish learning studies, including the effect of human handling versus that of experimenter-controlled USs. I conclude that systematic analyses of different USs are needed, along with detailed studies on how they may be optimized for the analysis of learning and memory in zebrafish.