A novel-odour exploration test for measuring anxiety in adult and larval zebrafish

Background: Existing methods to assess anxiety in zebrafish are mostly based on visual exploration. However, evidence suggests that zebrafish are more attuned to olfactory than visual stimuli. We developed a novel-odour exploration test (NOEt) for zebrafish. New Method: Adult zebrafish were exposed to a stimulus sponge soaked with olfactory cue and a control sponge with no cue, placed at the extremities of a narrow, rectangular tank. We scored time spent close to the two sponges to calculate the attraction towards the olfactory cue. In experiment 1, we tested adult zebrafish in the NOEt using various olfactory cues. In experiment 2, we tested larvae. In experiment 3, we compared the NOEt with the most used method to assess anxiety, the open-field test. Results: In experiment 1, zebrafish responded markedly to cues, by either approaching or avoiding the soaked sponge according to the type of cue. The temporal pattern of exploration toward the cue was similar to that of novel objects’ exploration in this species. In experiment 2, larvae responded to novel odours, but differently from adults. In experiment 3, we found a relationship between the NOEt and the open-field test. Comparison with Existing Method(s): Compared to existing tests, the NOEt has the advantage of exploiting the preferred sensory modality of zebrafish. Moreover, it can be used in early stages because olfactory receptors develop early in this species. Conclusions: The NOEt is a simple, rapid and low-cost test to study anxiety in zebrafish using the spontaneous exploration of novel olfactory cues.