Whole-Brain Mapping in Adult Zebrafish and Identification of the Functional Brain Network Underlying the Novel Tank Test

Zebrafish have gained prominence as a model organism in neuroscience over the past several decades, generating key insight into the development and functioning of the vertebrate brain. However, techniques for whole brain mapping in adult stage zebrafish are lacking. Here, we describe a pipeline built using open-source tools for whole-brain activity mapping in adult zebrafish. Our pipeline combines advances in histology, microscopy, and machine learning to capture <i>cfos</i> activity across the entirety of the brain. Following tissue clearing, whole brain images are captured using light-sheet microscopy and registered to the recently created adult zebrafish brain atlas (AZBA) for automated segmentation. By way of example, we used our pipeline to measure brain activity after zebrafish were subject to the novel tank test, one of the most widely used behaviors in adult zebrafish. <i>Cfos</i> levels peaked 15 minutes following behavior and several regions, including those containing serotoninergic and dopaminergic neurons, were active during exploration. Finally, we generated a novel tank test functional brain network. This revealed that several regions of the subpallium form a cohesive sub-network during exploration. Functional interconnections between the subpallium and other regions appear to be mediated primarily by ventral nucleus of the ventral telencephalon (Vv), the olfactory bulb, and the anterior part of the parvocellular preoptic nucleus (PPa). Taken together, our pipeline enables whole-brain activity mapping in adult zebrafish while providing insight into neural basis for the novel tank test.<b>Significance statement</b> Zebrafish have grown in popularity as a model organism over the past several decades due to their low cost, ease of genetic manipulation, and similarity to other vertebrates like humans and rodents. However, to date, tools for whole-brain mapping in adult stage animals has been lacking. Here, we present an open-source pipeline for whole-brain mapping in adult zebrafish. We demonstrate the use of our pipeline by generating a functional brain network for one of the most widely used behavioral assays in adult zebrafish, the novel tank test. We found that exploration of a novel tank engages the olfactory bulb and a network of subpallial regions that correspond to the mammalian subpallial amygdala and basal ganglia.