Abstract
Zebrafish are a widely utilised animal model in developmental genetics, and owing to recent advances in our understanding of zebrafish behaviour, their utility as a comparative model in behavioural neuroscience is beginning to be realised. One widely reported behavioural measure is the novel tank-diving assay, which has been often cited as a test of anxiety and stress reactivity. Despite its wide utilisation, and various validations against anxiolytic drugs, reporting of pre-test housing has been sparse in the literature. As zebrafish are a shoaling species, we predicted that housing environment would affect their stress reactivity and, as such, their response in the tank-diving procedure. In our first experiment, we tested various aspects of housing (large groups, large groups with no contact, paired, visual contact only, olfactory contact only) and found that the tank diving response was mediated by visual contact with conspecifics. We also tested the basal cortisol levels of group and individually housed fish, and found that individually housed individuals have lower basal cortisol levels. In our second experiment we found ethanol appeared to have an anxiolytic effect with individually housed fish but not those that were group housed. In our final experiment, we examined the effects of changing the fishes' water prior to tank diving as an additional acclimation procedure. We found that this had no effect on individually housed fish, but appeared to affect the typical tank diving responses of the group housed individuals. In conclusion, we demonstrate that housing represents an important factor in obtaining reliable data from this methodology, and should be considered by researchers interested in comparative models of anxiety in zebrafish in order to refine their approach and to increase the power in their experiments.
Highlights
Zebrafish (Danio rerio) are widely utilized as a developmental genetic model, and are fast becoming established as a model in behavioral neuroscience [1,2,3,4]
There was a reduction in time spent in the bottom third of the novel tank according to the level of grouping in the housing conditions, with the group housed fish showing the longest time on the bottom, and the individually housed fish spending the least time
This effect was confirmed with a linear mixed model (LMM) with time and group entered as fixed factors, ID nested in tank as random effects, and bottom duration as the response
Summary
Zebrafish (Danio rerio) are widely utilized as a developmental genetic model, and are fast becoming established as a model in behavioral neuroscience [1,2,3,4]. The utility of the species in this regard hinges on the implementation of behavioral assays with high construct validity and reliability [5]. The tank diving assay potentially fulfills this, and is commonly used as a measure of anxiety response to novel environments [9]. Similar to an open field procedure in rodents, anxiety is operationally defined by how much time the animal spends in the bottom half [10,11] or bottom third [12] of a novel tank, which is usually 70–85% of the first minute, and reducing thereafter (see [9] for a recent review). A variety of other behavioural markers (freezing, erratic swimming patterns) appear to be correlated with anxiety in the procedure (e.g., see [10,11]); it is not clear what aspects of anxiety these behaviours may represent as, to date, there is no convincing dissociation of any of the features either genetically, pharmacologically or otherwise [14]
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