DOI: 10.5061/dryad.k3j9kd5js
title: Whole-brain mapping in adult zebrafish and identification of the functional brain network underlying the novel tank test

description: 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 cfos 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. Cfos 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.
description: Please see methods in the associated paper
description: # Whole-brain mapping in adult zebrafish and identification of a novel
 tank test functional connectome
 [https://doi.org/10.5061/dryad.k3j9kd5js](https://doi.org/10.5061/dryad.k3j9kd5js) ## Description of the data and file structure See methods of associated paper for details on the creation of the dataset Files include: \***cfos_density_byregionand_timepoint.csv** Associated with figure 4 of the paper. This is the *cfos* cell density for individual fish for each brain region at each time point. Data fields * fish.id: internal reference for the individual fish * Sex: Sex of the animals (male or female) * Time point: Which time point the animals were euthanized, either off the housing rack (rack), after remaining in their hometank (Home tank) or time after exposure to the novel tank test (5, 15, 30, 60, or 120 minutes) * *Brain regions*: Abbreviations for brain regions; full region names are in Extended table 5-1 of the published manuscript and taken from the adult zebrafish brain atlas (azba.wayne.edu; Kenney et al, 2021) and are available in this repository in "Brain_region_names_and_ontological_levels.csv"  **Extended_table_5-2_15min_vs_HT_cell_counts.csv** Associated with figure 5 of the paper. This is the average counts per brain region for the home tank and fifteen minute groups, associated p-values from t-tests for each brain region, the effect size (Cohen's D) and the fdr corrected p-value. *Data fields* *IDX*: Region index number (from azba.wayne.edu; Kenney et al, 2021), can also be found in "Brain_region_names_and_ontological_levels.csv" *Abbreviation*: Abbreviation for brain region *Name*: Full name of brain region *Home.tank.mean*: Average *cfos* density for each brain region from the hometank fish *fifteen.minute.mean*: Average *cfos* density for each brain region from the group of fish euthanized fifteen minutes after the novel tank test *fold.change*: The fold increase in *cfos* density in the 15 minute group versus the hometank group *p.value*: The p-value from the independent samples t-test comparing the hometank to the 15 minute group *cohens.D*: The effect size (Cohen's D) for the comparison between the hometank and the 15 minute group *fdr*: False discovery rate (FDR) corrected p-values **15min_correlation_matrix.csv** Associated with figure 6 of the paper. Each row and column represent a brain region (abbreviated). Each entry is the correlated *cfos* activity between those brain regions across animals for the group of fish euthanized 15 minutes after the novel tank test. **Graph_15min.graphml and Graph_15min.cys** Associated with figure 7 of the paper. The 15 minute correlation with the top 2.5% of correlations to generate a network. In these network files, the nodes represent brain regions and the edges between nodes represent the presence of above threshold correlations. The .cys file is a Cytoscape session file that can be used to look at the network using Cytoscape ([https://cytoscape.org/](https://cytoscape.org/)). **Brain_region_names_and_ontological_levels.csv**\ List of brain regions, abbreviations, index numbers, and ontological levels. These correspond to the adult zebrafish brain atlas (AZBA) nomenclautre (azba.wayne.edu; Kenney et al , 2021).\ *Data fields*\ *IDX*: Region index number\ *Abbreviation*: The abbreviation for the brain region\ *Name*: The full name of the brain region\ *Ont.1-6*: The ontological levels for each region from most coarse/highest (1) to finest (2)\ *Display*: The ontological level used for display in figure 6 of the associated paper. **Bench_protocol_for_whole_brain_activity_mapping.docx**\ Detailed bench protocol for performing whole-brain activity mapping in adult zebrafish using iDISCO, CellFinder, and ANTs (advanced normalization tools) for registration to the adult zebrafish brain atlas.  **References**\ Kenney, J.W., Steadman, P.E., Young, O., Shi, M.T., Polanco, M., Dubaishi, S., Covert, K., Mueller, T. and Frankland, P.W., 2021. A 3D adult zebrafish brain atlas (AZBA) for the digital age. Elife, 10, p.e69988. ## Code/software **remove_overlaps.py**\ Python code for removing overlaps after CellFinder: **Kenney_Data_setup_cellfinder_registration.R**\ R code for combining the output of segmentation from ANTS and cell counts from CellFinder: